A Larval Zebrafish Model for Assessing Hypoxic‐Induced
            In Vivo
            Cardiomyocyte Damage: Time Course for Induction and Cardiac Output Recovery

Background: Timely thrombolytic therapy for acute ischemic stroke is important for improving neurological prognosis. Screening of thrombolytic agents on contemporary animal models of focal ischemic stroke is generally difficult as they require sophisticated surgical procedures such as induction of cerebral artery occlusion through mechanical ligation. Herein we seek to develop a novel zebrafish (Danio rerio) model of acute ischemic stroke for screening of thrombolytic drugs. Methods: All experiments were performed on a modified confocal optical microscope, which allows the induction of thrombosis at a selected blood vessel of larval zebrafish and the imaging of thrombotic and thrombolytic processes in real time. To initiate photochemical thrombosis, a 532-nm laser was focused at a blood vessel of a larva (4 dpf) that had been injected with a photosensitizer (rose bengal). To test the thrombolytic activity of tPA, we injected tPA to the blood vessel of a larval with a thrombus that partially occluded the blood flow. Results: Through photochemical means, we induced endothelial injury at selected blood vessel which subsequently triggered thrombosis. We show that an occlusion at the 1st branch of central artery drastically diminished the hemodynamics and cardiac function of larval zebrafish and impaired their capability to maintain balance during swimming whereas that at the basilar artery resulted in a high death rate. Immunofluorescent imaging shows that the photochemically induced thrombus comprised fibrins and platelets. After the injection of tPA to a larval with a partially occluded blood vessel, the fibrin mesh on the thrombus appeared sparse and limp which eventually led to the restoration of blood flow. Conclusions: Our zebrafish model of ischemic stroke is convenient to adapt and is highly reproducible. This model can potentially become an effective platform that benefits the screening of thrombolytic agents and the optimization of their dose.

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Kaempferol-3-O-Glucuronide Ameliorates Non-Alcoholic Steatohepatitis in High-Cholesterol-Diet-Induced Larval Zebrafish and HepG2 Cell Models via Regulating Oxidation Stress

Life ◽

10.3390/life11050445 ◽

2021 ◽

Vol 11 (5) ◽

pp. 445

Author(s):

Yang Deng ◽

Ji Ma ◽

Xin Weng ◽

Yuqin Wang ◽

Maoru Li ◽

...

Keyword(s):

Oxidative Stress ◽

Lipid Accumulation ◽

Hepg2 Cells ◽

High Cholesterol Diet ◽

Cholesterol Diet ◽

High Cholesterol ◽

Zebrafish Model ◽

Alcoholic Fatty Liver ◽

Larval Zebrafish

NAFLD (non-alcoholic fatty liver disease) is one of the most prominent liver diseases in the world. As a metabolic-related disease, the development of NAFLD is closely associated with various degrees of lipid accumulation, oxidation, inflammation, and fibrosis. Ilex chinensis Sims is a form of traditional Chinese medicine which is used to treat bronchitis, burns, pneumonia, ulceration, and chilblains. Kaempferol-3-O-glucuronide (K3O) is a natural chemical present in Ilex chinensis Sims. This study was designed to investigate the antioxidative, fat metabolism-regulating, and anti-inflammatory potential of K3O. A high-cholesterol diet (HCD) was used to establish steatosis in larval zebrafish, whereby 1mM free fatty acid (FFA) was used to induce lipid accumulation in HepG2 cells, while H2O2 was used to induce oxidative stress in HepG2. The results of this experiment showed that K3O reduced lipid accumulation and the level of reactive oxygen species (ROS) both in vivo (K3O, 40μM) and in vitro (K3O, 20μM). Additionally, K3O (40μM) reduced neutrophil aggregation in vivo. K3O (20μM) also decreased the level of malondialdehyde (MDA) and significantly increased the level of glutathione peroxidase (GSH-px) in both the HCD-induced larval zebrafish model and H2O2-exposed HepG2 cells. In the mechanism study, keap1, nrf2, tnf-α, and il-6 mRNA were all significantly reversed by K3O (20μM) in zebrafish. Changes in Keap1 and Nrf2 mRNA expression were also detected in H2O2-exposed HepG2 cells after they were treated with K3O (20μM). In conclusion, K3O exhibited a reduction in oxidative stress and lipid peroxidation, and this may be related to the Nrf2/Keap1 pathway in the NAFLD larval zebrafish model.

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BMAA and MCLR interact to modulate behavior and exacerbate molecular changes related to neurodegeneration in larval zebrafish

Toxicological Sciences ◽

10.1093/toxsci/kfaa178 ◽

2020 ◽

Author(s):

Rubia M Martin ◽

Michael S Bereman ◽

Kurt C Marsden

Keyword(s):

Acoustic Startle ◽

Fold Increase ◽

Health Concern ◽

P Value ◽

Dependent Manner ◽

Zebrafish Model ◽

Molecular Changes ◽

Larval Zebrafish ◽

Morphological Defects

Abstract Exposure to toxins produced by cyanobacteria (i.e., cyanotoxins) is an emerging health concern due to their increasing prevalence and previous associations with neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). The objective of this study was to evaluate the neurotoxic effects of a mixture of two co-occurring cyanotoxins, β-methylamino-L-alanine (BMAA) and microcystin leucine and arginine (MCLR), using the larval zebrafish model. We combined high-throughput behavior-based toxicity assays with discovery proteomic techniques to identify behavioral and molecular changes following 6 days of exposure. While neither toxin caused mortality, morphological defects, or altered general locomotor behavior in zebrafish larvae, both toxins increased acoustic startle sensitivity in a dose-dependent manner by at least 40% (p < 0.0001). Furthermore, startle sensitivity was enhanced by an additional 40% in larvae exposed to the BMAA/MCLR mixture relative to those exposed to the individual toxins. Supporting these behavioral results, our proteomic analysis revealed a 4-fold increase in the number of differentially expressed proteins (DEPs) in the mixture-exposed group. Additionally, prediction analysis reveals activation and/or inhibition of 8 enriched canonical pathways (enrichment p-value < 0.01; z-score ≥|2|), including ILK, Rho Family GTPase, RhoGDI, and calcium signaling pathways, which have been implicated in neurodegeneration. We also found that expression of TDP-43, of which cytoplasmic aggregates are a hallmark of ALS pathology, was significantly upregulated by 5.7-fold following BMAA/MCLR mixture exposure. Together, our results emphasize the importance of including mixtures of cyanotoxins when investigating the link between environmental cyanotoxins and neurodegeneration as we reveal that BMAA and MCLR interact in vivo to enhance neurotoxicity.

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The Cyanotoxin 2,4-DAB Reduces Viability and Causes Behavioral and Molecular Dysfunctions Associated with Neurodegeneration in Larval Zebrafish

Neurotoxicity Research ◽

10.1007/s12640-021-00465-4 ◽

2022 ◽

Author(s):

Rubia M. Martin ◽

Michael S. Bereman ◽

Kurt C. Marsden

Keyword(s):

Metabolic Pathways ◽

Protein Profile ◽

Acoustic Startle ◽

Motor Dysfunction ◽

Zebrafish Model ◽

Larval Zebrafish ◽

Neurotoxic Effects ◽

And Behavior ◽

Lateral Sclerosis

AbstractExposure to cyanotoxins has been linked to neurodegenerative diseases, including amyotrophic lateral sclerosis, Alzheimer’s, and Parkinson’s disease. While the cyanotoxin β-methylamino-L-alanine (BMAA) has received much attention, cyanobacteria produce many cyanotoxic compounds, several of which have been detected in nature alongside BMAA, including 2,4-diaminobutyric acid (2,4-DAB) and N-(2-aminoethyl)glycine (AEG). Thus, the question of whether 2,4-DAB and AEG also cause neurotoxic effects in vivo is of great interest, as is the question of whether they interact to enhance toxicity. Here, we evaluate the toxic and neurotoxic effects of these cyanotoxins alone or in combination by measuring zebrafish larval viability and behavior after exposure. 2,4-DAB was the most potent cyanotoxin as it decreased larval viability by approximately 50% at 6 days post fertilization, while BMAA and AEG decreased viability by just 16% and 8%, respectively. Although we only observed minor neurotoxic effects on spontaneous locomotion, BMAA and AEG enhanced acoustic startle sensitivity, and they interacted in an additive manner to exert their effects. 2,4-DAB; however, only modulated startle kinematics, an indication of motor dysfunction. To investigate the mechanisms of 2,4-DAB’s effects, we analyzed the protein profile of larval zebrafish exposed to 500 µM 2,4-DAB at two time points and identified molecular signatures consistent with neurodegeneration, including disruption of metabolic pathways and downregulation of the ALS-associated genes SOD1 and UBQLN4. Together, our data demonstrate that BMAA and its isomers AEG and 2,4-DAB cause neurotoxic effects in vivo, with 2,4-DAB as the most potent of the three in the zebrafish model.

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Sensory Neuron Sodium Current Requires Nongenomic Actions of Thyroid Hormone During Development

Journal of Neurophysiology ◽

10.1152/jn.90801.2008 ◽

2008 ◽

Vol 100 (5) ◽

pp. 2719-2725 ◽

Cited By ~ 26

Author(s):

Marc A. Yonkers ◽

Angeles B. Ribera

Keyword(s):

Thyroid Hormone ◽

Time Course ◽

Sodium Current ◽

Active Form ◽

Hormone Regulation ◽

Zebrafish Model ◽

Sodium Conductance ◽

Rapid Effect ◽

Touch Response

Development of the embryonic nervous system requires thyroid hormone. However, the underlying mechanisms and targets of thyroid hormone action are not well defined. To identify embryonic roles for thyroid hormone we tested for effects on a key neuronal trait, voltage-gated sodium current ( INa), in the zebrafish model system. We recorded from Rohon–Beard sensory neurons (RBs) using whole cell voltage-clamp methods. Here, we provide in vivo evidence for thyroid hormone regulation of INa. Chronic thyroid hormone application increased RB peak INa density 1.4-fold. However, INa density showed a similar increase within 5 min of an acute hormone application, a time course not expected for a genomic mechanism. Tetraiodothyroacetic acid (tetrac), a thyroid hormone blocker, blocked both chronic and acute effects. Further, the thyroid hormone precursor thyroxine (T4) affected INa, yet the traditionally active form triiodothyronine did not. Consequently, we tested for a nonconventional T4 receptor. LM609, a selective antagonist of integrin αVβ3, occluded the rapid effect of T4, implicating a specific integrin dimer as a T4 receptor. Chronic application of either tetrac or LM609 significantly reduced sodium conductance, demonstrating an in vivo requirement for T4-integrin regulation of INa. Further, removing endogenous T4 levels via yolkectomy reduced sodium conductance, an effect that was partially rescued by T4 supplementation following surgery. Because RBs mediate the embryonic touch response, we tested for behavioral effects. Tetrac and LM609 significantly reduced the percentage of touch trials eliciting a normal touch response. T4's rapid effect on RB INa highlights the importance of embryonic T4 availability and nongenomic T4 signaling.

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A transgenic zebrafish model of neutrophilic inflammation

Blood ◽

10.1182/blood-2006-05-024075 ◽

2006 ◽

Vol 108 (13) ◽

pp. 3976-3978 ◽

Cited By ~ 548

Author(s):

Stephen A. Renshaw ◽

Catherine A. Loynes ◽

Daniel M.I. Trushell ◽

Stone Elworthy ◽

Philip W. Ingham ◽

...

Keyword(s):

Time Course ◽

Digital Image Analysis ◽

Experimental Manipulation ◽

In Vivo Model ◽

Transgenic Zebrafish ◽

Zebrafish Model ◽

Similar Time ◽

Zebrafish Larvae ◽

Fluorescent Cells

Abstract We have established an in vivo model for genetic analysis of the inflammatory response by generating a transgenic zebrafish line that expresses GFP under the neutrophil-specific myeloperoxidase promoter. We show that inflammation is induced after transection of the tail of zebrafish larvae and that this inflammation subsequently resolves over a similar time course to mammalian systems. Quantitative data can be generated from this model by counting of fluorescent cells or by digital image analysis. In addition, we show that the resolution of experimentally induced inflammation can be inhibited by the addition of a pancaspase inhibitor, zVD.fmk, demonstrating that experimental manipulation of the resolution of inflammation is possible in this model.

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Exposure to a mixture of BMAA and MCLR synergistically modulates behavior in larval zebrafish while exacerbating molecular changes related to neurodegeneration

10.1101/2020.07.15.205617 ◽

2020 ◽

Author(s):

Rubia M. Martin ◽

Michael S. Bereman ◽

Kurt C. Marsden

Keyword(s):

Acoustic Startle ◽

Fold Increase ◽

Health Concern ◽

P Value ◽

Dependent Manner ◽

Zebrafish Model ◽

Molecular Changes ◽

Larval Zebrafish ◽

Morphological Defects

AbstractExposure to toxins produced by cyanobacteria (i.e., cyanotoxins) is an emerging health concern due to their increased occurrence and previous associations with neurodegenerative disease including amyotrophic lateral sclerosis (ALS). The objective of this study was to evaluate the neurotoxic effects of a mixture of two co-occurring cyanotoxins, β-methylamino-L-alanine (BMAA) and microcystin leucine and arginine (MCLR), using the larval zebrafish model. We combined high-throughput behavior based toxicity assays with discovery proteomic techniques to identify behavioral and molecular changes following 6 days of exposure. While neither toxin caused mortality, morphological defects, or altered general locomotor behavior in zebrafish larvae, both toxins increased acoustic startle sensitivity in a dose-dependent manner by at least 40% (p<0.0001). Furthermore, startle sensitivity was enhanced by an additional 40% in larvae exposed to the BMAA/MCLR mixture relative to those exposed to the individual toxins. Supporting these behavioral results, our proteomic analysis revealed a 4-fold increase in the number of differentially expressed proteins (DEPs) in the mixture exposed group. Additionally, prediction analysis reveals activation and/or inhibition of 8 enriched canonical pathways (enrichment p-value<0.01; z-score≥|2|), including ILK, Rho Family GTPase, RhoGDI, and calcium signaling pathways, which have been implicated in neurodegeneration. We also found that expression of TDP-43, of which cytoplasmic aggregates are a hallmark of ALS pathology, was significantly upregulated by 5.7-fold following BMAA/MCLR mixture exposure. Together, our results emphasize the importance of including mixtures of cyanotoxins when investigating the link between environmental cyanotoxins and neurodegeneration as we reveal that BMAA and MCLR interact in vivo to enhance neurotoxicity.

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A Comprehensive Study of High Cholesterol Diet-Induced Larval Zebrafish Model: A Short-Time In Vivo Screening Method for Non-Alcoholic Fatty Liver Disease Drugs

International Journal of Biological Sciences ◽

10.7150/ijbs.30013 ◽

2019 ◽

Vol 15 (5) ◽

pp. 973-983 ◽

Cited By ~ 7

Author(s):

Ji Ma ◽

Hongli Yin ◽

Maoru Li ◽

Yang Deng ◽

Owais Ahmad ◽

...

Keyword(s):

Screening Method ◽

High Cholesterol Diet ◽

High Cholesterol ◽

Zebrafish Model ◽

Alcoholic Fatty Liver ◽

Larval Zebrafish ◽

Short Time ◽

Alcoholic Fatty Liver Disease ◽

Comprehensive Study

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Efficacy of Voriconazole against Aspergillus fumigatus Infection Depends on Host Immune Function

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00917-19 ◽

2019 ◽

Vol 64 (2) ◽

Cited By ~ 1

Author(s):

Emily E. Rosowski ◽

Jiaye He ◽

Jan Huisken ◽

Nancy P. Keller ◽

Anna Huttenlocher

Keyword(s):

Aspergillus Fumigatus ◽

Fungal Growth ◽

Fungal Spore ◽

Antifungal Drugs ◽

Zebrafish Model ◽

Content Type ◽

Larval Zebrafish ◽

Number Of Patients

ABSTRACT Antifungal therapy can fail in a remarkable number of patients with invasive fungal disease, resulting in significant morbidity worldwide. A major contributor to this failure is that while these drugs have high potency in vitro, we do not fully understand how they work inside infected hosts. Here, we used a transparent larval zebrafish model of Aspergillus fumigatus infection amenable to real-time imaging of invasive disease as an in vivo intermediate vertebrate model to investigate the efficacy and mechanism of the antifungal drug voriconazole. We found that the ability of voriconazole to protect against A. fumigatus infection depends on host innate immune cells and, specifically, on the presence of macrophages. While voriconazole inhibits fungal spore germination and growth in vitro, it does not do so in larval zebrafish. Instead, live imaging of whole, intact larvae over a multiday course of infection revealed that macrophages slow down initial fungal growth, allowing voriconazole time to target and kill A. fumigatus hyphae postgermination. These findings shed light on how antifungal drugs such as voriconazole may synergize with the immune response in living hosts.

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The cyanotoxin 2,4-DAB enhances mortality and causes behavioral and molecular dysfunctions associated with neurodegeneration in larval zebrafish

10.1101/2021.10.13.464292 ◽

2021 ◽

Author(s):

Rubia M. Martin ◽

Michael S. Bereman ◽

Kurt C. Marsden

Keyword(s):

Metabolic Pathways ◽

Protein Profile ◽

Acoustic Startle ◽

Motor Dysfunction ◽

Zebrafish Model ◽

Larval Zebrafish ◽

Neurotoxic Effects ◽

And Behavior ◽

Lateral Sclerosis

Exposure to cyanotoxins has been linked to neurodegenerative diseases, including amyotrophic lateral sclerosis, Alzheimer's, and Parkinson's disease. While the cyanotoxin beta-methylamino-L-alanine (BMAA) has received much attention, cyanobacteria produce many cyanotoxic compounds, several of which have been detected in nature alongside BMAA including 2,4-diaminobutyric acid (2,4-DAB), and N-(2-aminoethyl)glycine (AEG). Thus, the question of whether DAB and AEG also cause neurotoxic effects in vivo is of great interest, as is the question of whether they interact to enhance toxicity. Here, we evaluate the toxic and neurotoxic effects of these cyanotoxins alone or in combination by measuring zebrafish larval viability and behavior after exposure. 2,4-DAB was the most potent cyanotoxin as it decreased larval viability by approximately 50% at 6 days post fertilization, while BMAA and AEG decreased viability by just 16% and 8%, respectively. Although we only observed minor neurotoxic effects on spontaneous locomotion, BMAA and AEG enhanced acoustic startle sensitivity, and they interacted in an additive manner to exert their effects. 2,4-DAB, however, only modulated the startle kinematics, an indication of motor dysfunction. To investigate the mechanisms of 2,4-DAB's effects, we analyzed the protein profile of larval zebrafish exposed to 500 uM 2,4-DAB at two time points and identified molecular signatures consistent with neurodegeneration, including disruption of metabolic pathways and downregulation of the ALS-associated genes SOD1 and UBQLN4. Together, our data demonstrate that BMAA and its isomers AEG and 2,4-DAB cause neurotoxic effects in vivo, with 2,4-DAB as the most potent of the three in the zebrafish model.

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