Transgenic Zebrafish Research Articles

Investigation of Kelussia Odoratissima and Angelica Sinensis Similarities in Zebrafish-based In-vivo Bioactivity Assays and Their Chemical Composition.

Kelussia odoratissima and Angelica sinensis are two medicinal plants commonly used in Iran and China, respectively. They have been used in their indigenous traditional medicine, for various diseases including, blood refining, inflammation, cold, flu, stress, cardiovascular diseases, and nervous disorders. This study was conducted to evaluate the volatile oil composition of K. odoratissima leaves (KVL) and A. sinensis root (AVR); we also examined the biological activity of essential oils (EOs) and hydroalcoholic extracts of both plants using two different transgenic zebrafish (Danio rerio) models: angiogenesis and pancreatic beta cell (pBC) regeneration models. Both EOs were isolated by hydrodistillation and analysed by GC and GC/MS. For viability tests, larvae were treated with different concentrations of extracts to determine an appropriate starting concentration. Hydroalcoholic extracts and EOs have been tested in a dose-dependent manner for their biological activity using tissue-specific transgenic zebrafish Tg(fli-1: EGFP) and Tg (ins: GFP-NTR) embryos and larvae. One-way ANOVA was used to compare the mean of pBC area and intersegmental vessels (ISVs) outgrowth between the treatment groups. Eleven compounds were in common to both oils, comprising 51.3% of KVL and 61.7% of AVR, of which 39.3% in KVL and 37.6% in AVR were phthalide structures. Results revealed that both EOs blocked ISVs formation in the Tg (fli-1: EGFP) embryos increased to 10% of the control value, while both hydroalcoholic extracts did not show any anti-angiogenesis effects in these embryos. In addition, AVR has been shown to significantly induce PBC regeneration following ablation in the Tg (ins: GFP-NTR), but its regenerative activity was lower than that of 5'-N-ethylcarboxamidoadenosine (NECA) as a positive control. Taken together, the anti-angiogenesis activity of both EOs could be attributed to the phthalide structures while for the PBC regenerative activity, other compounds including β-Thujaplicinol, exclusively existing in AVR, might be effective. Although the genera, organs, and origin of these plants are different, their similar chemical composition and biological activities make them valuable resources for further investigation in basic medical and pharmaceutical science.

4-Methylbenzylidene camphor triggers estrogenic effects via the brain-liver-gonad axis in zebrafish larvae

4-Methylbenzylidene camphor (4-MBC), an emerging contaminant, is a widely-used ultraviolet (UV) filter incorporated into cosmetics because it protects the skin from UV rays and counters photo-oxidation. Despite the well-established estrogenic activity of 4-MBC, the link between this activity and its effects on neurobehavior and the liver remains unknown. Thus, we exposed zebrafish larvae to environmentally relevant concentrations of 4-MBC with 1.39, 4.17, 12.5 and 15.4 μg/mL from 3 to 5 days postfertilization. We found that 4-MBC produced an estrogenic effect by intensifying fluorescence in the transgenic zebrafish, which was counteracted by co-exposure with estrogen receptor antagonist. 4-MBC-upregulated estrogen receptor alpha (erα) mRNA, and an interaction between 4-MBC and ERα suggested ERα′s involvement in the 4-MBC-induced estrogenic activity. RNA sequencing unearthed 4-MBC-triggered responses in estrogen stimulus and lipid metabolism. Additionally, 4-MBC-induced hypoactivity and behavioral phenotypes were dependent on the estrogen receptor (ER) pathway. This may have been associated with the disruption of acetylcholinesterase and acetylcholine activities. As a result, 4-MBC increased vitellogenin expression and caused lipid accumulation in the liver of zebrafish larvae. Collectively, this is the first study to report 4-MBC-caused estrogenic effects through the brain-liver-gonad axis. It provides novel insight into how 4-MBC perturbs the brain and liver development.

Intraocular Axon Regeneration in a Model of Penetrating Eye Injury.

Purpose: Penetrating eye injuries commonly cause permanent loss of vision in patients. Unlike mammals, zebrafish can regenerate both damaged tissue and severed axons in the central nervous system. Here, we present a tractable adult zebrafish model to study intraocular axon regeneration after penetrating eye injury. Methods: To create consistent penetrating intraocular injuries, pins of standardized diameters were inserted into the eye through the cornea and penetrating the retina but not the underlying sclera. Transgenic gap43:GFP reporter fish were used to preferentially label retinal ganglion cells (RGCs) that respond to injury with regenerating axons. Retinas were fixed and flat mounted at various times postinjury to examine injury size, number of green fluorescent protein (GFP)-positive cells and axons, axonal varicosities, and rate of regeneration to the optic nerve head. Intraocular injection of colchicine was used to inhibit axon outgrow as a proof of principle that this method can be used to screen effects of pharmacological agents on intraocular axon regeneration. Results: Penetrating injury to the zebrafish retina results in robust axon regeneration by RGCs around and beyond the site of injury. The gap43:GFP transgene allows visualization of individual or small bundles of axons with varicosities and growth cones easily observable. Regeneration proceeded with most, if not all, axons reaching the optic nerve head by 3-day postinjury. A single intraocular injection of colchicine a day after injury was sufficient to delay axon regeneration at 2-days postinjury. Surprisingly, we identified a stereotypically located population of circumferential projecting neurons within the retina that upregulate gap43:GFP expression after injury. Conclusions: Penetrating injury to the adult gap43:GFP transgenic zebrafish eye is a model of successful intraocular axon regeneration. The pharmacological and genetic tools available for this organism should make it a powerful tool for dissecting the cellular, molecular, and genetic mechanisms of axon regeneration in the intraocular environment.

Azithromycin inhibits glioblastoma angiogenesis in mice via inducing mitochondrial dysfunction and oxidative stress.

The poor outcomes in glioblastoma (GBM) necessitate new treatments. As GBM is highly vascularized and its growth is largely dependent on angiogenesis, angiogenesis inhibitors have been hotly evaluated in clinical trials for GBM treatment for the last decade. In line with these efforts, our work reveals that azithromycin, a clinically available antibiotic, is a novel angiogenesis inhibitor. Azithromycin inhibits vessel structure formation on Matrigel of GBM-derived endothelial cell (ECs) and other types of ECs. Time course analysis shows that azithromycin interferes with the early stage of angiogenesis. Azithromycin also inhibits GBM-derived EC adhesion, growth and survival but not migration. The transgenic zebrafish Tg (fli1a: EGFP) model clearly shows that azithromycin inhibits angiogenesis in vivo. Of note, azithromycin at non-toxic dose inhibits GBM growth in mice and increases overall survival, and furthermore, this is associated with angiogenesis inhibition. Mechanism studies show that azithromycin decreases mitochondrial respiration by suppressing the activity of multiple complexes, leading to ATP reduction, oxidative stress and damage. In addition, oxidative stress induced by azithromycin is through thiol redox-mediated pathways. Our work demonstrates the anti-angiogenic activity of azithromycin via inducing mitochondrial dysfunction and oxidative stress. Our pre-clinical evidence provides a rationale for initiating clinical trials using azithromycin in combination with standard-of-care drugs for GBM patients.

Human Mutated MYOT and CRYAB Genes Cause a Myopathic Phenotype in Zebrafish.

Myofibrillar myopathies (MFMs) are a group of hereditary neuromuscular disorders sharing common histological features, such as myofibrillar derangement, Z-disk disintegration, and the accumulation of degradation products into protein aggregates. They are caused by mutations in several genes that encode either structural proteins or molecular chaperones. Nevertheless, the mechanisms by which mutated genes result in protein aggregation are still unknown. To unveil the role of myotilin and αB-crystallin in the pathogenesis of MFM, we injected zebrafish fertilized eggs at the one-cell stage with expression plasmids harboring cDNA sequences of human wildtype or mutated MYOT (p.Ser95Ile) and human wildtype or mutated CRYAB (p.Gly154Ser). We evaluated the effects on fish survival, motor behavior, muscle structure and development. We found that transgenic zebrafish showed morphological defects that were more severe in those overexpressing mutant genes. which developed a myopathic phenotype consistent with that of human myofibrillar myopathy, including the formation of protein aggregates. Results indicate that pathogenic mutations in myotilin and αB-crystallin genes associated with MFM cause a structural and functional impairment of the skeletal muscle in zebrafish, thereby making this non-mammalian organism a powerful model to dissect disease pathogenesis and find possible druggable targets.

Development of a Transparent Transgenic Zebrafish Cellular Phenotype Tg(6xNF-kB:EGFP); Casper(roy-/-, nacre-/-) to Study NF-kB Activity.

NF-κB signaling has broad effects on cell survival, tissue growth, and proliferation activities. It controls many genes that are involved in inflammation and thus is a key player in many inflammatory diseases. The elevation of NF-κB activators is associated with elevated mortality, especially in cancer and cardiovascular diseases. The zebrafish has emerged as an important model for whole-organism in vivo modeling in translational research. In vertebrates, in-vivo spatial resolution is limited due to normal opacification of skin and subdermal structure. For in vivo imaging, skin transparency by blocking the pigmentation via chemical inhibition is required and the maintenance of this transparency is vital. The Casper(roy-/-, nacre-/-) mutant of zebrafish maintains this transparency throughout its life and serves as an ideal combination of sensitivity and resolution for in vivo stem cell analyses and imaging. We developed an NF-kB:GFP/Casper transparent transgenic zebrafish cellular phenotype to study inflammatory processes in vivo. We outline the experimental setup to generate a transparent transgenic NF-kB/Casper strain of zebrafish through the cross-breeding of Casper and NF-kB transgenic adult fish and have generated F01 in the form of heterozygous progeny. The transgenic F01 progeny was further inbred to generate heterozygous progenies from F1 to F4 generations. Furthermore, it continued to successfully develop the homozygous strain Tg(6xNF-kB:EGFP); Casper(roy-/-, nacre-/-) in the F05 generation. This novel strain of F05 generation showed 100% homozygosity in the transgenic transparent progeny of Tg(6xNF-kB:EGFP); Casper(roy-/-, nacre-/-). The strain has been confirmed by generating the F06 generation of homozygous progeny and again verified and validated for its homogeneity in the F07 generation. The newly developed novel transparent transgenic strain of the NF-kB reporter line has been coined as "Tg(6xNF-kB:EGFP); Casper(roy-/-, nacre-/-)gmc1". We have established a newly generated phenotype of transparent transgenic zebrafish for time-lapse in vivo confocal microscopy to study the cellular phenotype and pathologies at the cellular level over time. This will allow for quantifying the changes in the NF-kB functional activities over time and allow the comparison of control and cardiac-oncology experimental therapeutics. We validated the newly developed Tg(6xNF-kB:EGFP); Casper(roy-/-, nacre-/-)gmc1 homozygous strain of zebrafish by studying the inflammatory response to bacterial lipopolysaccharide (LPS) exposure, tolerance, and the inhibitory role of a potential novel drug candidate against LPS-induced inflammation. The results establish the unique application of newly developed strains by identifying hit and lead drug candidates for experimental therapeutics.

Dynamic and broad expression of adamts9 in developing and adult zebrafish.

Previous studies showed that Adamts9 is involved in multiple functions including ovulation, spine formation, primordial germ cell migration, and development of primary ovarian follicles in animals. However, systemic examination and high-resolution analyses of adamts9 expression are missing due to lack of a sensitive reporter assay. In the present study, we created a new transgenic zebrafish reporter line Tg(adamts9:EGFP) and assayed its expression in various tissues and cells during development and in adults at high-resolution using confocal imaging. Reporter expression was validated with real-time quantitative PCR, whole mount in situ hybridization, and immunohistochemistry for endogenous adamts9. Strong expression of the adamts9:EGFP transgene was found in a wide range of adult and embryonic zebrafish tissues/cells including ovaries, testes, brains, eyes, pectoral fins, intestine, skin, gill, muscle, and heart; while lower expression was observed in the liver and growing ovarian follicles (stages II and III). Our results of a broad and dynamic expression pattern for this evolutionary conserved metalloprotease suggest involvement of adamts9 in the development and physiological functions of various tissues in animals.

Loss of gdnfa disrupts spermiogenesis and male courtship behavior in zebrafish

Spermatogenesis is essential for establishment and maintenance of reproduction in male vertebrates. Spermatogenesis, which is mainly regulated by the combined action of hormones, growth factors, and epigenetic factors, is highly conserved. Glial cell line-derived neurotrophic factor (GDNF) is a member of the transforming growth factor-β superfamily. In this study, global gdnfa knockout and Tg (gdnfa: mcherry) transgenic zebrafish lines were generated. Loss of gdnfa resulted in disorganized testes, decreased gonadosomatic index, and low percentage of mature spermatozoa. In the Tg (gdnfa: mcherry) zebrafish line, we found that gdnfa was expressed in Leydig cells. The mutation in gdnfa significantly decreased Leydig cell marker gene expression and androgen secretion in Leydig cells. In addition, courtship behavior was disrupted in the male mutants. We present in vivo data showing that global knockout of gdnfa disrupts spermiogenesis and male courtship behavior in zebrafish. The first viable vertebrate model with a global gdnfa knockout may be valuable for studying the role of GDNF in animal reproduction.

Acute immune responses in zebrafish and evasive behavior of a parasite - who is winning?

The protozoan parasite Ichthyophthirius multifiliis is an economically important parasite for the aquaculture- and ornamental fish industry. The parasite is abundant worldwide and infects the skin, gills and fins of freshwater fish species. For approximately the last fifty years the innate and protective immune mechanisms induced by I. multifiliis have been in focus in different fish hosts. By utilizing transgenic zebrafish, new tools to investigate this have emerged. The aim of this study was therefore to elucidate early immune responses in zebrafish larvae by using gene expression and in vivo imaging of neutrophil and macrophage behavior during infection. For the first time, zebrafish larvae were infected with the parasite and infection dynamics, parasite size and host-parasite interactions were investigated. Results showed that the larvae responded with mild inflammation and that the 12 compared to 5 days post fertilization larvae were significantly less susceptible. It was furthermore observed that neutrophils and macrophages were attracted to the parasites and that neutrophils reacted with neutrophil extracellular traps (NETs) when fighting the parasite. The parasite was rotating vigorously, presumably to impede the neutrophils and macrophages from attaching to it but on rare occasions, neutrophils and macrophages were able to kill the parasite. Based on these observations, we concluded that the parasite uses the rotation as an immune evasive strategy and that the zebrafish larvae respond with high activity from neutrophils and macrophages locally but systemically only with mild inflammation.

1761-P: Verapamil Increases ß-Cells Proliferation in MIN6 Cells and ß-Cell Mass in Zebrafish Model

Recent clinical studies highlighted a significant clinical utility for verapamil in both type 1 (T1D) and type 2 (T2D) diabetes. Verapamil treatment in T1D lowered dependency on exogenous insulin. In T2D, addition of verapamil to metformin therapy improved glycemic control. These findings led us to further study the molecular and cellular mechanisms of verapamil in the treatment of T1D and T2D using an in vitro (MIN6 mouse β cell-line) and in vivo zebrafish (Danio rerio) model. In this study, MIN6 cells were cultured in growth media containing 5.6 mM glucose (mimicking normoglycemia) and supplemented with 2% BSA to eliminate any proliferative effect of high glucose in the media, and growth factors in FBS. The cells were treated with different concentrations of verapamil. The levels of cell proliferation (MTT assay), cell growth (cell counts), Ki67 expression (IF), and cell cycle (flow cytometry) were studied in MIN6 cells treated with the optimal dose detected (50 μM) of verapamil. Larvae (24hpf) of the Ins:NfsB-mCherry transgenic zebrafish (expressing mCherry fluorescence reporter under the control of insulin proximal promoter) were treated with 10 uM of verapamil for 3 days. Full larvae images were acquired using a stereo microscope. Our results show that verapamil induced significantly increased proliferation of MIN6 cells. Cell cycle analysis shows initiation of the proliferative effect during an early time point (1-4 hours) after verapamil treatment. Higher Ki67 expression indicated enhanced cellular proliferation at 24 hours and growth curve data confirmed that the proliferative effect of verapamil lasted for 7 days. As evident from increased mCherry fluorescence intensity in zebrafish larvae, verapamil treatment increased the β cell mass during the exposure period of three days. Taken together, our findings support the positive impact of verapamil on mouse β cell proliferation in vitro as well as on increased β cell mass in Zebrafish model. Disclosure H.Arefanian: None. S.E.John: None. S.Jacob: None. M.Alhusayan: None. S.J.Kurian: None. S.P.Kochumon: None. S.T.K.Sindhu: None. F.Alzaid: None. M.Abu-farha: None. J.Abubaker: None. A.T.Thangavel: None. A.Al madhoun: None. R.Ahmad: None. F.Almulla: None. F.Alrashed: None. F.Bahman: None. H.Alsaeed: None. N.A.Abukhalaf: None. S.Shenouda: None. M.R.Williams: None. R.Nizam: None. Funding Kuwait Foundation for the Advancement of Sciences (RACB-2019-001)

Terbutryn causes developmental toxicity in zebrafish (Danio rerio) via apoptosis and major organ malformation in the early stages of embryogenesis

Terbutryn (2-(ethylamino)-4-(tert-butylamino)-6-(methylthio)-1,3,5-triazine) is a substituted symmetrical triazine herbicide used in agricultural fields to prevent undesired vegetation growth by inhibiting photosynthesis in target weeds. Although terbutryn has various benefits, long-term exposure, misuse, or abuse of terbutryn may cause non-target toxicity and severe ecosystem pollution. To provide a detailed description of the embryonic developmental toxicity of terbutryn, zebrafish (Danio rerio) were exposed to 2, 4, and 6 mg/L of terbutryn and the morphological changes, pathological abnormalities, and developmental endpoints were assessed relative to that of a solvent control. The results showed that terbutryn induces a loss of survivability, reduction in body and eye size, and edema in the yolk sac. Through fluorescence microscopy, blood vessels, motor neurons, and liver development were investigated using transgenic zebrafish models based on fluorescently tagged genes (fllk1:eGFP, olig2:dsRed, and L-fabp:dsRed). Furthermore, cell death by apoptosis in zebrafish caused by terbutryn exposure was evaluated via acridine orange staining, which is a selective fluorescent staining agent. To support the preceding results, gene expression alterations caused by terbutryn exposure in zebrafish larvae were assessed. The overall results indicate that exposure to terbutryn induces apoptosis and disrupts organ development. These embryonic developmental toxicity results suggest that terbutryn should be applied in the right areas at the appropriate rates, concentrations, and quantities.

Method for Visualization of Emergency Granulopoiesis in the Zebrafish Embryo.

Emergency granulopoiesis (EG) is a response to severe inflammation in which increased neutrophils are generated in the hematopoietic tissue. Photolabeling is utilized to distinguish newly developed neutrophils from existing neutrophils. However, this technique requires a strong laser line and labels subsets of the existing neutrophils. Here we create a transgenic zebrafish line that expresses a time-dependent switch from green fluorescent protein (GFP) to red fluorescent protein (RFP) in neutrophils, which allows quantification of EG using simple GFP/RFP ratiometric imaging.

MDB-42. UNCOVERING DYNAMIC CHANGES IN MEDULLOBLASTOMA ASSOCIATED VASCULATURE IN ZEBRAFISH

Abstract Medulloblastoma (MB) is an embryonal-derived lesion arising in the cerebellum, contributing to 20% of childhood brain tumours and 63% of intracranial embryonal tumours. Currently, standard of care includes surgical resection and chemotherapy, used in conjunction with radiation. While these treatments have significantly improved survival rates, the therapy side effects are detrimental to survivors’ quality of life. Furthermore, relapse occurs in 30% of patients and unfortunately these are untreatable and therefore fatal for 95% of patients. It is presumed that in patients that present with a recurrent tumour, initial treatment has been ineffective. A major cause of this can be the heterogenous vasculature in and around the tumour. Recent studies have identified that the blood brain barrier (BBB), in the context of MB, is highly heterogenous with differences in BBB cellular composition and integrity between MB sub-types and within single tumours. It remains unclear however, how these vascular differences emerge and what the primary defects are. This gap in our knowledge is mainly due to the lack of models that allow long-term live imaging. We have established a xenograft approach to examine human MB tumour cells in zebrafish embryonal brains. We have validated that human Medulloblastoma cells of the Group 3 (Gp3) MB subgroup, are viable in zebrafish brains. This work has also identified that in the presence of these human Gp3 MB tumour cells, the local vasculature becomes dysmorphic and tortuous over time. Timelapse imaging of these tumour and vasculature interactions demonstrates that there is a rapid and long ranging angiogenic response with new vessel sprouts growing towards the tumour cells. By utilising our large range of transgenic zebrafish lines, we will continue investigating how MB cells interact and alter distinctive BBB cell types and how these changes impact vessel function.

Fluchloralin induces developmental toxicity in heart, liver, and nervous system during early zebrafish embryogenesis

The zebrafish is a prominent vertebrate model popularly used for toxicity testing because of its rapid development and transparent embryos. Fluchloralin, a dinitroaniline herbicide used to control weeds, inhibits microtubule formation and cell division. The structurally homologous substances ethalfluralin and pendimethalin, which belong to the dinitroaniline family, were found to be genotoxic and to exert developmental toxicity via mitochondrial dysfunction in a zebrafish model. To date, developmental toxicity of fluchloralin in zebrafish has not been reported. In the present study, we identified morphological changes in developing zebrafish, including decreased survival rate and body length, and increased yolk sac edema. In dose-dependent response to fluchloralin exposure, inhibition of neurogenesis in the spinal cord and motor neuron defects were observed in transgenic zebrafish models (olig2:dsRed). Zebrafish exposed to fluchloralin also displayed organ dysfunction in the heart, liver, and pancreas in cmlc2:dsRed and lfabp:dsRed;elastase:GFP transgenic models. Fluchloralin increased cell death in the brain by promoting apoptosis, visualized via acridine orange staining, and by activating apoptosis signaling proteins, including cytochrome c1, zBax, and Bcl-XL. This study provides novel evidence supporting the necessity of controlling pollutants in aquatic environments.

Zebrafish thrombosis models according to the location of thrombus formation.

Ischemic stroke becomes a major cause of death and disability. It can develop due to intravascular or cardiac thromboemboli. Animal models that reflect diverse stroke mechanisms remain under development. Using photochemical thrombosis, we developed a feasible zebrafish model according to the thrombus location (intracerebral vs. intracardiac). We validated the model using real-time imaging and thrombolytic agent. We used transgenic zebrafish larvae (flk:gfp), which express specific fluorescence in endothelial cells. We injected Rose Bengal, a photosensitizer as a mixture of photosensitizer, and a fluorescent agent into the cardinal vein of the larvae. We then evaluated real-time thrombosis in vivo by inducing thrombosis through exposure to a confocal laser (560 nm) and staining the blood flow (RITC-dextran). We validated intracerebral and intracardiac thrombotic models with checking the activity of tissue plasminogen activator (tPA). The photochemical agent induced the formation of intracerebral thrombi in transgenic zebrafish. Real-time imaging techniques confirmed the formation of the thrombi. The damage and apoptosis of the vessel's endothelial cells were seen in the in vivo model. An intracardiac thrombosis model was developed by the same method using photothrombosis, and the model was validated through thrombolysis by tPA. We developed and validated two zebrafish thrombosis models that are readily available, cost-effective, and intuitive for assessing the efficacy of thrombolytic agents. These models can be used for a broad spectrum of future studies, such as screening and efficacy assessment of new antithrombotic agents.

Paroxetine induced larva zebrafish cardiotoxicity through inflammation response

Paroxetine (PRX) is a common antidepressant drug which widely existence in natural environment. Numerous studies in the past few decades have focused on the beneficial effects of PRX on depression, however, the toxic properties and the potential mechanisms remain unclear. In this study, zebrafish embryos were exposed to 1.0, 5.0, 10 and 20 mg/L of PRX from 4 to 120-hour-post-fertilization (hpf), and it showed that PRX exposure caused adverse effects in zebrafish embryos, including decreased body length, blood flow velocity, cardiac frequency, cardiac output and increased burst activity and atria area. Meanwhile, the Tg (myl7: EGFP) and Tg (lyz: DsRed) transgenic zebrafish were used to detect the cardiotoxicity and inflammation response of PRX. Moreover, the heart development associated genes (vmhc, amhc, hand2, nkx2.5, ta, tbx6, tbx16 and tbx20) and inflammatory genes (IL-10, IL-1β, IL-8 and TNF-α) were up-regulated after PRX challenge. In addition, Aspirin was used to alleviate the PRX-induced heart development disorder. In conclusion, our study verified the PRX induced inflammatory related cardiotoxicity in larva zebrafish. Meanwhile, the current study shown the toxic effects of PRX in aquatic organism, and provide for the environmental safety of PRX.

Analysis of the mechanism of action of preoperative bilirubin that causes cognitive impairment after liver transplantation: verification of permeability of high-concentration bilirubin

Background: We previously found an association between preoperative bilirubin concentration and cognitive impairment after liver transplantation. In order to study a possible mechanism for bilirubin causing postoperative cognitive dysfunction, we investigated the effect of high concentrations of bilirubin on the blood–brain barrier. Methods: Our model system was 30-week-old transgenic zebrafish fabp10a:cfp-NTR. In preliminary experiments we confirmed systemic absorption after retro-orbital injection using reddish Rhodamine-dextran injected with a Hamilton syringe. For the hyperbilirubinaemia model we used retro-orbital injection of bilirubin, with subsequent measurement of bilirubin concentration in the brain and blood. Food reward learning was conducted for 4 days using the T-maze, and learning cognitive ability videos were filmed without food reward on the 5th day (control group=9/bilirubin group=9). Again using Fabp10a:cfp-NTR zebrafish, methimazole was injected to damage the liver, and after 3 and 7 days, the brain was extracted and stained with Evans blue to check the blood–brain barrier permeability. Results: In the T-maze test, there was no difference between the bilirubin and control groups (P=0.4135). There was no statistically significant difference in the bilirubin level measured in the brain after retro-orbital injection of bilirubin on the 1st, 2nd, and 3rd days between the 10-fold and 50-fold injection groups. However, bilirubin measured in blood decreased over time as blood concentrations increased. As a result of Evans blue staining, there was no difference in colour between the 3rd and 7th day brains compared with the control (Fig. 9). Conclusion: As a result of this study, cognitive dysfunction occurs when liver damage occurs, but this seems to occur even without a change in blood–brain barrier permeability, and bilirubin is not considered to be the sole cause of cognitive dysfunction.

The influence of size and surface chemistry on the bioavailability, tissue distribution and toxicity of gold nanoparticles in zebrafish (Danio rerio)

Gold nanoparticles (AuNPs) are widely used in biomedicine and their specific properties including, size, geometrics, and surface coating, will affect their fate and behaviour in biological systems. These properties are well studied for their intended biological targets, but there is a lack of understanding on the mechanisms by which AuNPs interact in non-target organisms when they enter the environment. We investigated the effects of size and surface chemistry of AuNPs on their bioavailability, tissue distribution and potential toxicity using zebrafish (Danio rerio) as an experimental model. Larval zebrafish were exposed to fluorescently tagged AuNPs of different sizes (10–100 nm) and surface modifications (TNFα, NHS/PAMAM and PEG), and uptake, tissue distribution and depuration rates were measured using selective-plane illumination microscopy (SPIM). The gut and pronephric tubules were found to contain detectable levels of AuNPs, and the concentration-dependent accumulation was related to the particle size. Surface addition of PEG and TNFα appeared to enhance particle accumulation in the pronephric tubules compared to uncoated particles. Depuration studies showed a gradual removal of particles from the gut and pronephric tubules, although fluorescence indicating the presence of the AuNPs remained in the pronephros 96 h after exposure. Toxicity assessment using two transgenic zebrafish reporter lines, however, revealed no AuNP-related renal injury or cellular oxidative stress. Collectively, our data show that AuNPs used in medical applications across the size range 40–80 nm, are bioavailable to larval zebrafish and some may persist in renal tissue, although their presence did not result in measurable toxicity with respect to pronephric organ function or cellular oxidative stress for short term exposures.

Hydrogen peroxide signaling modulates neuronal differentiation via microglial polarization and Wnt/β-catenin pathway.

Reactive oxygen species (ROS) are generated within the cell and serve as second messengers in fundamental cellular processes under physiologic conditions. Although the deleterious effects of high-level ROS associated with oxidative stress are well established, it is unclear how the developing brain reacts to redox changes. Our aim is to investigate how redox alteration affects neurogenesis and the mechanism that underlies it.We investigated in vivo microglial polarization and neurogenesis in zebrafish after hydrogen peroxide (H2O2) incubation. To quantify intracellular H2O2 levels in vivo, a transgenic zebrafish line that expresses Hyper and termed Tg(actb2:hyper3)ka8 was used. Then, in vitro studies with N9 microglial cells, 3-dimensional neural stem cell (NSC)-microglia coculture, and conditioned medium experiments are carried out to comprehend the mechanism underlying the changes in neurogenesis upon redox modulation.In zebrafish, exposure to H2O2 altered embryonic neurogenesis, induced M1 polarization in microglia, and triggered the Wnt/β-catenin pathway. N9 microglial cell culture experiments revealed that exposure to H2O2 resulted in M1 polarization in microglial cells, and this polarization was mediated by the Wnt/β-catenin pathway. Redox modulation of microglia interfered with NSC differentiation in coculture experiments. Neuronal differentiation was significantly higher in NSCs cocultured with H2O2-treated microglia when compared to control microglia. Wnt inhibition prevented the effects of H2O2-treated microglia on NSCs. No significant alterations were observed in conditioned medium experiments.Our findings point to a robust interplay between microglia and neural progenitors influenced by the redox state. Intracellular H2O2 levels can interfere with neurogenesis by altering the phenotypic state of the microglia via the Wnt/β-catenin system.

An Efficient and Reproducible Method for the Isolation and Culture of Primary Cardiomyocytes from Adult Zebrafish.

Zebrafish is a popular animal model in regeneration studies due to their ability to regenerate the heart. Primary cardiomyocytes could be an alternative tool for studying the intrinsic mechanisms of cardiovascular disease in vitro. Thus, our objective is to develop an efficient protocol to isolate primary cardiomyocytes from zebrafish hearts. Low concentration of digestive enzyme (0.5 mg/mL collagenase type II) was utilized in our protocol to obtain single-cell suspension. The ventricles were fragmented, mechanically pipetted, and constantly shaken to ensure adequate contact between the tissues and the enzyme. Preplating the cell suspension onto culture plates for 2 h helped remove cardiac fibroblasts. The purity of isolated cells was validated by flow cytometry analysis of transgenic zebrafish with cardiomyocyte-specific expression of enhanced green fluorescent protein (EGFP) or endothelial cell-specific expression of mCherry. Quantitative real-time PCR analysis revealed a high level of the purity, with cardiac fibroblasts, endothelial cells, and epicardial cell markers scarcely detected in the purified cells. Altogether, this study established a reproducible protocol for isolating primary cardiomyocytes with high purity and activity from adult zebrafish hearts that can be cultured in vitro for up to 4 weeks. This protocol provides a valuable tool for studying the intrinsic mechanisms of cardiovascular disease in vitro using primary cardiomyocytes.