AB Strain Research Articles

Comparison of baseline cataract rates in AB and TL wildtype zebrafish strains

Zebrafish are an outstanding model for assessing the involvement of genes in paediatric cataracts. Gene discovery for cataracts is enhanced by manipulation of the genome of zebrafish embryos and comparing the phenotypes of mutant progeny with the wildtype embryos. However, wildtype laboratory fish can also develop cataracts, potentially confounding the results. In this study, we compared the baseline cataract rate between two commonly used wildtype laboratory strains, AB and TL, and also an outbred transgenic line with mCherry reporter. We assessed a total of 805 lens images of fish at 4 days post-fertilisation for cataracts and scored each cataract observed as mild, moderate or severe. We found that the AB strain had a cataract rate of 16.2%, TL had 8.9%, and mCherry had 0.7% and these rates were significantly different. We found that TL strain had a lower rate of mild cataracts than AB fish, however, the rate of moderate and severe phenotypes in the AB and the TL strain was similar. Overall, we showed that the baseline cataract rate varies significantly between the strains housed in a single facility and conclude that baseline rates of cataracts should be assessed when planning experiments to assess the genetic causes of cataracts.

Establishment of a zebrafish inbred strain, M-AB, capable of regular breeding and genetic manipulation

Inbred strains of organisms are genetically highly uniform and thus useful for life science research. We have previously reported the ongoing generation of the zebrafish IM strain from the India (IND) strain through full sib-pair mating for 16 generations. However, the IM fish laid a small number of offspring and had a short lifespan, implying the need for discreet care in breeding. Here, we report the subsequent establishment of IM strain as well as the generation of a new inbred zebrafish strain, Mishima-AB (M-AB). M-AB was derived from the *AB strain by full sib-pair mating for over 20 generations, which fulfills the general criterion for the establishment of an inbred strain. In contrast to the IM case, maintenance of the M-AB strain by sib-pair mating required almost no special handling. Genome sequencing of IM individuals from the 47th generation and M-AB individuals from the 27th generation revealed that SNP-based genomic heterogeneity across whole-genome nucleotides was 0.008% and 0.011%, respectively. These percentages were much lower than those of the parental IND (0.197%) and *AB (0.086%) strains. These results indicate that the genomes of these inbred strains were highly homogenous. We also demonstrated the successful microinjection of antisense morpholinos, CRISPR/Cas9, and foreign genes into M-AB embryos at the 1-cell stage. Overall, we report the establishment of a zebrafish inbred strain, M-AB, which is capable of regular breeding and genetic manipulation. This strain will be useful for the analysis of genetically susceptible phenotypes such as behaviors, microbiome features and drug susceptibility.

Azospirilla and Nitrogen Fertilization to Promote Maize Seed Development and Yield

Aims: This study evaluated the interaction of Azospirillum brasilense (Ab) strains and nitrogen fertilizer doses on maize.
 Study Design: Laboratory experiment evaluated Ab strains (PCR and PTF) on maize seed germination and vigor (completely randomized design). Field experiment evaluated Ab strains and nitrogen fertilization on maize yield parameters (randomized block design).
 Place and Duration of Study: The germination and vigor experiment (10 days in April 2019) was implemented in a controlled environment at the Seed Analysis Laboratory of the Universidade Federal de Uberlândia, Brazil. The field experiment (137 days in winter 2019) was implemented at the Fisioplant Research Station (Uberlândia, Brazil) under commercial maize crop management.
 Methodology: In controlled conditions (laboratory), 200 seeds of maize (K9105VIP3, Riber KWS Seeds) were incubated (25 °C) with the Ab strain solutions for germination and vigor evaluations. In the field, 60,000 plants ha-1 (0.5 m space between sowing lines) of maize were cropped under combinations of nitrogen fertilizer and volumes of Ab strains (treatments) for leaf nitrogen, SPAD index, leaf count, fresh and dry maize shoot biomass, one-thousand grains weight, grain rows per maize ear, productivity (kg ha-1).
 Results: Treatment with 100 mL ha-1 of Ab PFT strain increased maize seed germination and vigor more than Ab PCR strain and control (no-inoculation or fertilizer). The number of grain rows of the 100% N + 150 mL ha-1 of Ab PFT strain and the maize shoot dry mass of the 100% N (no-inoculation) were higher than the control. The treatments did not affect the SPAD index, weight of one thousand grains, fresh maize shoot biomass, or productivity.
 Conclusion: Using Ab with nitrogen fertilizer is a promising strategy to improve maize crop responses; however, more studies are needed to detail the ideal management of Ab strains for maize crops and yield construction.

Direct male development in chromosomally ZZ zebrafish.

The genetics of sex determination varies across taxa, sometimes even within a species. Major domesticated strains of zebrafish (Danio rerio), including AB and TU, lack a strong genetic sex determining locus, but strains more recently derived from nature, like Nadia (NA), possess a ZZ male/ZW female chromosomal sex-determination system. AB fish pass through a juvenile ovary stage, forming oocytes that survive in fish that become females but die in fish that become males. To understand mechanisms of gonad development in NA zebrafish, we studied histology and single cell transcriptomics in developing ZZ and ZW fish. ZW fish developed oocytes by 22 days post-fertilization (dpf) but ZZ fish directly formed testes, avoiding a juvenile ovary phase. Gonads of some ZW and WW fish, however, developed oocytes that died as the gonad became a testis, mimicking AB fish, suggesting that the gynogenetically derived AB strain is chromosomally WW. Single-cell RNA-seq of 19dpf gonads showed similar cell types in ZZ and ZW fish, including germ cells, precursors of gonadal support cells, steroidogenic cells, interstitial/stromal cells, and immune cells, consistent with a bipotential juvenile gonad. In contrast, scRNA-seq of 30dpf gonads revealed that cells in ZZ gonads had transcriptomes characteristic of testicular Sertoli, Leydig, and germ cells while ZW gonads had granulosa cells, theca cells, and developing oocytes. Hematopoietic and vascular cells were similar in both sex genotypes. These results show that juvenile NA zebrafish initially develop a bipotential gonad; that a factor on the NA W chromosome, or fewer than two Z chromosomes, is essential to initiate oocyte development; and without the W factor, or with two Z doses, NA gonads develop directly into testes without passing through the juvenile ovary stage. Sex determination in AB and TU strains mimics NA ZW and WW zebrafish, suggesting loss of the Z chromosome during domestication. Genetic analysis of the NA strain will facilitate our understanding of the evolution of sex determination mechanisms.

The Therapeutic Effects and Mechanisms of Gardenoside on Inflammatory Injury via the INS/NF-κB Pathway in a T2DM Zebrafish Model

Objective: This study utilized the zebrafish model to investigate the therapeutic effects of gardenoside on inflammatory damage in type 2 diabetes mellitus (T2DM) through the INS/NF-κB pathway. Methods: The study employed 7-8-month-old AB strain zebrafish and induced T2DM models by continuous exposure to a 2% glucose solution for 14 days. Control, model, gardenoside low-, medium-, and high-dose groups (2.5 mg/L, 5 mg/L, 10 mg/L), and a metformin group were established. Parameters such as zebrafish body weight, fasting blood glucose levels, and behavioral analyses were monitored. Fructosamine levels were measured using ELISA, while real-time polymerase chain reaction (Real-time PCR) was employed to assess the relative expression levels of INS, IRS, NF-κB, IL-1β, and IL-6 genes. Intestinal histological examination was conducted to observe inflammation levels. Results: Compared to the control group, the model group exhibited significantly increased blood glucose levels, weight gain, and fructosamine content. High-speed locomotion time increased, while low-speed locomotion time decreased. Relative expression levels of INS, NF-κB, IL-1β, and IL-6 genes were elevated (P < 0.0001), whereas IRS relative expression levels decreased (P E0.001). In comparison to the model group, the gardenoside and metformin groups demonstrated reduced blood glucose levels and no significant change in body weight. The gardenoside low and medium dose groups, as well as the metformin group, showed reduced high-speed locomotion time and increased low-speed locomotion time. The relative expression levels of INS, NFκB, IL-1β, and IL-6 genes in the gardenoside and metformin groups decreased (P < 0.01 or P < 0.0001), while IRS relative expression levels increased (P < 0.001 or P < 0.0001). Conclusion: Gardenoside may suppress inflammatory responses, alleviate insulin resistance, and ameliorate diabetes-related inflammatory symptoms, potentially through the regulation of the INS/NF-κB signaling pathway.

Foxl2l is a germ cell-intrinsic gatekeeper of oogenesis in zebrafish.

Zebrafish serve as a valuable model organism for studying germ cell biology and reproductive processes. The AB strain of zebrafish is proposed to exhibit a polygenic sex determination system, where most males initially develop juvenile ovaries before committing to male fate. In species with chromosomal sex determination, gonadal somatic cells are recognized as key determinants of germ cell fate. Notably, the loss of germ cells in zebrafish leads to masculinization, implying that germ cells harbor an intrinsic feminization signal. However, the specific signal triggering oogenesis in zebrafish remains unclear. In the present study, we identified foxl2l as an oocyte progenitor-specific gene essential for initiating oogenesis in germ cells. Results showed that foxl2l-knockout zebrafish bypassed the juvenile ovary stage and exclusively developed into fertile males. Further analysis revealed that loss of foxl2l hindered the initiation of oocyte-specific meiosis and prevented entry into oogenesis, leading to premature spermatogenesis during early gonadal development. Furthermore, while mutation of the pro-male gene dmrt1 led to fertile female differentiation, simultaneous disruption of foxl2l in dmrt1 mutants completely blocked oogenesis, with a large proportion of germ cells arrested as germline stem cells, highlighting the crucial role of foxl2l in oogenesis. Overall, this study highlights the unique function of foxl2l as a germ cell-intrinsic gatekeeper of oogenesis in zebrafish.

Molecular basis of retinal remodeling in a zebrafish model of retinitis pigmentosa

A hallmark of inherited retinal degenerative diseases such as retinitis pigmentosa (RP) is progressive structural and functional remodeling of the remaining retinal cells as photoreceptors degenerate. Extensive remodeling of the retina stands as a barrier for the successful implementation of strategies to restore vision. To understand the molecular basis of remodeling, we performed analyses of single-cell transcriptome data from adult zebrafish retina of wild type AB strain (WT) and a P23H mutant rhodopsin transgenic model of RP with continuous degeneration and regeneration. Retinas from both female and male fish were pooled to generate each library, combining data from both sexes. We provide a benchmark atlas of retinal cell type transcriptomes in zebrafish and insight into how each retinal cell type is affected in the P23H model. Oxidative stress is found throughout the retina, with increases in reliance on oxidative metabolism and glycolysis in the affected rods as well as cones, bipolar cells, and retinal ganglion cells. There is also transcriptional evidence for widespread synaptic remodeling and enhancement of glutamatergic transmission in the inner retina. Notably, changes in circadian rhythm regulation are detected in cones, bipolar cells, and retinal pigmented epithelium. We also identify the transcriptomic signatures of retinal progenitor cells and newly formed rods essential for the regenerative process. This comprehensive transcriptomic analysis provides a molecular road map to understand how the retina remodels in the context of chronic retinal degeneration with ongoing regeneration.

Биологические свойства штаммов L. helveticus

The biological properties of three strains of L. helvetcus were studied. All studied strains metabolize D-galactose. The L. helveticus AB strain utilizes D-glucose, D-fructose, N-acetylglucosamine, and D-trehalose; L. helveticus 20T — D-mannose, N-acetylglucosamine; L. helveticus H-9 — D-glucose, D-lactose, D-trehalose. All strains showed high β-galactosidase activity, as well as the activity of acid phosphatase, naphthol-AS-BI hydrolase, leucine arylamidase, and cystine arylamidase. For L. helveticus 20T and L. helveticus AB strains, valine arylamidase activity was found. All strains are sensitive to gentamicin, ampicillin, tetracycline, azithromycin, lincomycin, chloramphenicol. The data obtained indicate the safety of using strains as part of starter cultures and as producers of metabolite additives in terms of reducing the risk of spreading antibiotic resistance.

The Biocompatibility of a Ginger-Containing Herbal Toothpaste on Developing Zebrafish Embryos

The biocompatibility of toothpaste in an oral cavity should be approved by clinical trials. Nowadays, herbal toothpaste is increasing in popularity due to its natural ingredients. Being genetically similar to humans, zebrafish are used in potential toxicity testing. The zebrafish embryotoxicity test is a fast and straightforward method to study chemical toxicity during embryogenesis. Objective: This study aimed to evaluate if there was any biocompatibility of the toothpaste on zebrafish embryos. Methods: Adult AB strain zebrafish were used according to Institutional Animal Care and Use Committee protocols. Normally dividing, spherical embryos were exposed to herbal toothpaste with ginger (Gumgumix, Turkey) (50 mg/L) and conventional toothpaste (Signal, Expert Protection, Bulgaria) (50 mg/L) in well plates containing 20 embryos, having four replicates. Developmental effects, mortality, and hatching rates were evaluated for 72h. Results: Zebrafish embryos exposed to conventional toothpaste had a higher mortality rate than those exposed to herbal toothpaste; they hatched later and delayed in development. There was no difference between herbal toothpaste and the control group regarding mortality and hatching rates (p > 0.005). Conclusion: The herbal toothpaste showed higher biocompatibility on zebrafish embryos compared to the conventional toothpaste under the condition of this study.

Tris(1,3-dichloro-2-propyl) phosphate causes female-biased growth inhibition in zebrafish: Linked with gut microbiota dysbiosis

Tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) is ubiquitous in aquatic environment, but its effect on intestinal health of fish has yet not been investigated. In the present study, the AB strain zebrafish embryos were exposed to environmentally realistic concentrations (0, 30, 300, and 3000 ng·L−1) of TDCIPP for 90 days, after which the fish growth and physiological activities were evaluated, and the intestinal microbes were analyzed by 16S rRNA gene high-throughput sequencing. Our results manifested that the body length and body weight were significantly reduced in the female zebrafish but not in males. Further analyses revealed that TDCIPP resulted in notable histological injury of intestine, which was accompanied by impairment of epithelial barrier integrity (decreased tight junction protein 2), inflammation responses (increased interleukin 1β), and disruption of neurotransmission (increased serotonin) in female intestine. Male intestines maintained intact intestinal structure, and the remarkably increased activity of glutathione peroxidase (GPx) might protect the male zebrafish from inflammation and intestinal damage. Furthermore, 16S rRNA sequencing analysis showed that TDCIPP significantly altered the microbial communities in the intestine in a gender-specific manner, with a remarkable increase in alpha diversity of the gut microbiome in male zebrafish, which might be another mechanism for male fish to protect their intestines from damage by TDCIPP. Correlation analysis revealed that abnormal abundances of pathogenic bacteria (Chryseobacterium, Enterococcus, and Legionella) might be partially responsible for the impaired epithelial barrier integrity and inhibition in female zebrafish growth. Taken together, our study for the first time demonstrates the high susceptibility of intestinal health and gut microbiota of zebrafish to TDCIPP, especially for female zebrafish, which could be partially responsible for the female-biased growth inhibition.

Lantana camara leaf extract ameliorates memory deficit and the neuroinflammation associated with scopolamine-induced Alzheimer’s-like cognitive impairment in zebrafish and mice

Context Lantana camara Linn. (Verbenaceae) is used for improving memory in certain African societies. Objective This study investigated the effect of prophylactic treatment with hydroethanolic leaf extract of Lantana camara (LCE) on short-term memory deficit and neuroinflammation induced with scopolamine in zebrafish and mice. Materials and methods Zebrafish (AB strain) and mice (ICR) were given donepezil (0.65 mg/kg, oral) and LCE (10, 30, 100 mg/kg, oral) for 7, and 10 days, respectively, before induction of cognitive impairment with scopolamine immersion (200 µM) and intraperitoneal injection (2 mg/kg), respectively. Spatial short-term memory was assessed in zebrafish using both Y- and T-mazes, whereas Y-maze was used in mice. Mice hippocampal and cortical tissues were analyzed for mRNA expression of proinflammatory genes (IL-1β, IL-6, TNF-α, COX-2) using qRT-PCR. Results In the zebrafish Y-maze, LCE (10 and 100 mg/kg) increased time spent in the novel arm by 55.89 ± 5.70%, and 68.21 ± 2.75%, respectively, but not at 30 mg/kg. In the zebrafish T-maze, there was an increase in time spent in the food-containing arm at 30 (44.23 ± 2.13) and 100 mg/kg (52.30 ± 1.94). In the mouse Y-maze, spontaneous alternation increased by 52.89 ± 4.98% at only 10 mg/kg. LCE (10, 30, 100 mg/kg) inhibited proinflammatory gene (IL-1β, IL-6, TNF-α, COX-2) mRNA expression, with the highest inhibitory effect on IL-6 in both the hippocampus (83.27 ± 2.49%; 100 mg/kg) and the cortex (98.74 ± 0.11%; 10 mg/kg). Discussion and conclusion LCE ameliorated scopolamine-induced AD in both zebrafish and mice.

Different housing conditions for zebrafish: What are the effects?

Zebrafish is a popular experimental model in several research areas but little is known about the effects of using different strains or housing conditions. Poor control of genetic background and housing conditions could affect experimental results and data reproducibility. Here we investigated the effects of two possible sources of variation on zebrafish behaviour: fish origin and environmental parameters (light intensity, water temperature and noise). Zebrafish behaviour was then examined using the ‘novel tank test’, one of the most common paradigms used to assess anxiety-like behaviours in zebrafish. Our results show that an increase in light intensity alters fish behaviour, particularly freezing duration and distance from the bottom of the tank, indicating increased anxiety. Swimming activity increased at the lowest temperature (25 °C). However, different levels of background noise did not cause any significant changes in behaviour. Differences were also found between zebrafish strains and populations: while the AB strain from laboratory 1 was minimally influenced by variation in holding conditions, the AB strain from laboratory 2 was highly affected by changes in temperature, light, and background noise. Our study shows that variation in strains and holding conditions can significantly influence the results of behavioural testing and should be carefully considered in the experimental design and properly reported to improve data interpretation and reproducibility.

Beneficial Effect of Vitamin D on Non-Alcoholic Fatty Liver Disease (NAFLD) Progression in the Zebrafish Model

A major cause of chronic liver disease, cirrhosis, and hepatocellular carcinoma, non-alcoholic fatty liver disease (NAFLD) results from excessive liver fat accumulation. Vitamin D (VitD) plays multiple important roles in diverse physiologic processes. Here, we describe the role of VitD in the complex pathogenesis of NAFLD and explore the possible therapeutic role of VitD supplementation in NAFLD therapy. To compare the effect of VitD to other interventions such as low-calorie diet, we induced NAFLD in young adult zebrafish (Danio rerio, AB strain) and monitored the effects of VitD supplementation on the disease course. The zebrafish administered with high-dose VitD (1.25 μg) had significantly reduced liver fat compared to those that received low-dose VitD (0.049 μg) or caloric restriction. Gene expression analysis revealed that VitD downregulated several pathways that may play a role in NAFLD etiology, which affected fatty acid metabolism, vitamins and their cofactors, ethanol oxidation, and glycolysis. The pathway analysis revealed that the cholesterol biosynthesis pathway and the isoprenoid biosynthetic process pathway were significantly upregulated whereas the small molecule catabolic process pathway significantly downregulated following the exposure of NAFLD zebrafish model to high VitD dose. Therefore, our findings suggest the association of novel biochemical pathways with NAFLD and highlight the potential of VitD supplementation to reverse the severity of NAFLD, especially in younger people.

Dual-Toxin ("Bivalent") Infant Botulism in California, 1976-2020: Epidemiologic, Clinical, and Laboratory Aspects.

To assess the consequences of infant botulismthat result from Clostridium botulinum strains that produce 2 botulinum toxin serotypes, termed "bivalent." Epidemiologic investigations used a standard questionnaire. Clostridium botulinum strains were isolated by standard methods. Botulinum neurotoxin (BoNT) serotypes and the relative amounts of toxins produced were identified using the standard mouse bioassay. BoNT subtypes and genomic locations were identified by DNA nucleotide sequencing. Thirty bivalent cases of infant botulism occurred in the 45years (1976-2020), representing 2.0% of all California infant botulism cases, in the 3 geographic regions of southern California, the southern Central Valley, and mid-northern California. Toxin serotype combinations were Ba (n=22), Bf (n=7), and Ab (n=1). More patients with illness caused by bivalent C botulinum Ba and Bf strains needed endotracheal intubation at hospital admission, 60.0% (18/30), than did patients with illness caused by monovalent BoNT/B strains, 34.3% (152/443). The Cbotulinum Ba and Bf strains produced BoNT/B5 and either BoNT/A4 or /F2. The Ab strain produced BoNT/A2 and /B1. All toxin gene clusters were on plasmids. Infant botulism caused by bivalent Cbotulinum strains occurs sporadically and in diverse locations in California. Affected patients with bivalent Ba and Bf strains lacked distinguishing epidemiological features but appeared to be more severely paralyzed at hospital presentation than patients with illness caused by only BoNT/B. These bivalent strains produced BoNT subtypes A2, A4, B1, B5, and F2, and all toxin gene clusters were on plasmids.

Environmentally-relevant concentrations of the antipsychotic drugs sulpiride and clozapine induce abnormal dopamine and serotonin signaling in zebrafish brain

The presence of drugs in surface and groundwaters adversely affects the physiological function of non-target organisms due special activities that can pose a serious threats to various forms of aquatic life. Psychotropic drugs are one of the most commonly used drugs in the world. Hence, the aim of this study was to investigate the effect of environmentally-relevant concentrations of the antipsychotic drugs, sulpiride and clozapine, on dopaminergic (DAergic) and serotonergic (5-HTergic) neurotransmitter systems in the brain of zebrafish. Adult zebrafish (AB strain) were exposed to the environmentally-relevant concentrations of sulpiride, clozapine, or a mixture of sulpiride and clozapine. The effects of the drugs on the mRNA and protein levels of major functional molecules in DAergic and 5-HTergic systems were then analyzed in the telencephalon and diencephalon. Both drugs induced abnormal mRNA and protein levels of important functional molecules of the DA and 5-HT signaling pathways in both telencephalon and diencephalon, as shown by the abnormal transcriptional levels of TH, DAT, DR D1, DR D2, MAO, TPH, serotonin transporter (SERT), 5-HTR 1AA, 5-HTR 1B, 5-THR 2AA, and 5-HTR 2B, and the abnormal translational levels of DAT, DR D2, SERT, 5-HTR 1A, 5-HTR 1B, and 5-HTR 2B. In addition, we observed a specificity in the adverse effects of these antipsychotic drugs, in terms of doses and brain parts. Compared to their effects alone, the drug mixture had a weaker effect on the DA and 5-HT systems, suggesting an antagonistic interaction between sulpiride and clozapine. Our findings suggest that sulpiride and clozapine interfere with DAergic and 5-HTergic neurotransmitter systems in the telencephalon and diencephalon of zebrafish, resulting in possible effects on brain functions and posing a serious threat to the health of zebrafish.

Construction of zebrafish models for screening intracranial hemorrhage associated genes

Objective: To construct zebrafish models for the screening of intracranial hemorrhage (ICH) associated genes. Methods: ICH zebrafish models were constructed through morpholino oligonucleotides (MOs) technique and microinjection technique, and multiple verification was performed from macro and micro perspectives. First, the normal wild-type AB strain zebrafish injected with control MO was used as the control group, and AB zebrafish embryos microinjected with MOs of genes related to development of neural crest-derived cells (NCDCs) were used as the study group, such as col8a1 MO, tfap2α MO, msx1a MO, msx2 MO, and dkk1a MO. Preliminary verification of the model was conducted under a white-light optical microscope. Then, the model was verified by Tg (flk1: gfp; gata1: dsRed) double transgenic zebrafish, with vascular endothelial cells labeled by green fluorescent protein (GFP) and red blood cell labeled by fluorescent protein (dsRed), and thus the location of cerebral hemorrhage can be observed more clearly. Specifically, zebrafish embryos were microinjected with Control MO as the control group and those microinjected with col8a1 MO as the study group. Then the embryos were cultured until 48 hours post-fertilization to observe the leakage of red blood cells under the confocal laser scanning microscope. Finally, Tg (flk1: gfp) transgenic zebrafish was used to verify the model based on the blood-brain barrier (BBB). Through the leakage of dextran-rhodamine and DAPI dyes, the destruction of BBB and the occurrence of cerebral hemorrhage in zebrafish were further clarified, and quantitative statistics were carried out to verify the relationship between NCDCs development related genes and cerebral hemorrhage phenotype, which proved that the modeling was effective. Results: The zebrafish with col8a1, tfap2α, and msx1 mutations in the study group had apparent ICH compared with wildtype zebrafish, and the prevalence of ICH was 18.18% (52/286), 23.04% (62/251), and 35.94% (23/64), respectively. While, the zebrafish with msx2 and dkk1a mutations rarely had ICH, with the ICH prevalence of 1.03% (1/97) and 1.15% (1/87), respectively. The prevalence of red blood cells leakage in Tg (flk1:gfp; gata1:dsred) double transgenic zebrafish injected with Control Mo and col8a1 Mo was 0.37% (1/273) and 18.18% (52/286) (P<0.001). The number of DAPI positive nuclei of Tg (flk1: gfp) transgenic zebrafish injected with Control Mo and col8a1 Mo was 10.05±5.27 and 60.35±3.96 (P<0.001), and the fluorescent intensity of midbrain parenchymal induced by dextran-rhodamin leakage was 2.54±4.70 and 5.13±3.52 (P<0.001). Conclusion: This study successfully constructs the ICH zebrafish models, and ICH-related genes are screened out, such as col8a1, tfap2α, msx1, and so on.

Metabolic Competency of Larval Zebrafish in Drug-Induced Liver Injury: A Case Study of Acetaminophen Poisoning.

Larval zebrafish is emerging as a new model organism for studying drug-induced liver injury (DILI) with superiorities in visual assessment, genetic engineering as well as high throughput. Metabolic bioactivation to form reactive intermediates is a common event that triggers DILI. This study first addressed the correlation between acetaminophen metabolism and hepatotoxicity in zebrafish larvae (3-day postfertilization) and demonstrated the occurrence of cytochrome P450 enzymes-mediated acetaminophen (APAP) bioactivation at early developmental stage through characterizing the dose-effect (0-1.6 mg/ml) and the time course (0-72 h) of liver injury and metabolism in the AB strain and LiPan transgenic line Tg(lfabp10a: DsRed; elaA:egfp) expressing the liver-specific fluorescent protein. APAP caused multiorgan developmental retardation and elicited dose- and time-dependent hepatotoxicity. Liver imaging revealed significant changes earlier than histological and biochemical measurements. APAP bioactivation in larval zebrafish was first confirmed by the detection of the glutathione conjugate of the reactive intermediate NAPQI (NAPQI-GSH) and subsequent mercapturate derivatives NAPQI-cysteine and NAPQI-N-acetylcysteine after even short (0.5-h postexposure) or low (0.2 mg/ml) APAP exposure. APAP overdose impaired metabolic function, in particular sulfation, whereas facilitated GSH depletion and APAP sulfate excretion. Meanwhile, APAP displayed triphasic accumulation in the larvae, agreeing with fluctuating metabolic capabilities with sulfation dominating the early larval developmental stage. Most importantly, the dose-response effects and time course of APAP accumulation and metabolism agree well with those of the liver injury development. Overall, larval zebrafish has developed mammalian-like metabolic function, enabling it an ideal model organism for high-throughput screening hepatotoxicity and mechanistic study of bioactivation-based DILI.

Boldness in Zebrafish Larvae—Development and Differences between a Domesticated Lab Strain and Offspring of Wild-Caught Fish

Zebrafish (Danio rerio) are becoming one of the most important model organisms in behavioural neuroscience. It has been shown repeatedly that different zebrafish strains show large behavioural differences. These divergent behavioural profiles may have a genetic basis, but environmental factors and previous experience are also known to greatly affect the behavioural phenotype of zebrafish. It could be expected that behavioural differences at the larval stage should be less affected by environmental factors and experience. In the present study, we screened larvae of zebrafish of the AB strain and offspring of wild-caught zebrafish for boldness, using an open field test. In order to follow the behavioural development, we studied larvae at the age of 5-, 7-, 12- and 30-days post fertilization (dpf). Behaviour, as well as behavioural development, clearly differed between the larvae of the different strains. Wild larvae showed larger total distance moved than AB larvae, both at light and dark conditions. These differences were already present at 12 dpf but became more pronounced with age. Wild larvae had a greater variance compared to AB larvae for most of the variables. We have previously shown that bold and shy adult zebrafish differ in the brain expression of dopamine and opioid receptors. The results of the current study show that wild larvae display significantly higher brain expression of drd2b than AB larvae at 30 dpf, a difference that could be related to differences in activity. We did not detect any differences in the expression of opioid receptors.

The Relationship between Procyanidin Structure and Their Protective Effect in a Parkinson's Disease Model.

This study evaluated the effect of grape seed-derived monomer, dimeric, and trimeric procyanidins on rat pheochromocytoma cell line (PC12) cells and in a zebrafish Parkinson’s disease (PD) model. PC12 cells were cultured with grape seed-derived procyanidins or deprenyl for 24 h and then exposed to 1.5 mm 1-methyl-4-phenylpyridinium (MPP+) for 24 h. Zebrafish larvae (AB strain) 3 days post-fertilization were incubated with deprenyl or grape seed-derived procyanidins in 400 µM 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) for 4 days. The results showed that the procyanidin dimers procyanidin B1 (B1), procyanidin B2 (B2), procyanidin B3 (B3), procyanidin B4 (B4), procyanidin B1-3-O-gallate (B1-G), procyanidin B2-3-O-gallate (B2-G), and the procyanidin trimer procyanidin C1 (C1) had a protective effect on PC12 cells, decreasing the damaged dopaminergic neurons and motor impairment in zebrafish. In PC12 cells and the zebrafish PD model, procyanidin (B1, B2, B3, B4, B1-G, B2-G, C1) treatment decreased the content of reactive oxygen species (ROS) and malondialdehyde (MDA), increased the activity of antioxidant enzymes glutathione peroxidase (GSH-Px), catalase (CAT), and superoxide dismutase (SOD), and upregulated the expression of nuclear factor-erythroid 2-related factor (Nrf2), NAD(P)H: quinone oxidoreductase 1 (NQO1), and heme oxygenase-1 (HO-1). These results suggest that in PC12 cells and the zebrafish PD model, the neuroprotective effects of the procyanidins were positively correlated with their degree of polymerization.

Tetramethylpyrazine Protects Endothelial Injury and Antithrombosis via Antioxidant and Antiapoptosis in HUVECs and Zebrafish

Chuanxiong Rhizoma, the dried rhizome of Ligusticum chuanxiong Hort., is a commonly used drug for promoting blood circulation and dissipating congestion. Tetramethylpyrazine (TMP), the main active ingredient of Ligusticum chuanxiong, has significant antioxidant, anti-inflammatory, and vascular protective effects. However, the protective properties and underlying mechanisms of TMP against endothelial injury-induced insufficient angiogenesis and thrombosis have not been elucidated. Therefore, we aimed to explore the protective effects of TMP on endothelial injury and its antithrombotic effects and study the mechanism. In vitro experiments showed that TMP could alleviate hydrogen peroxide– (H2O2–) induced endothelial injury of human umbilical vein endothelial cells (HUVECs) and the protective mechanism might be related to the regulation of MAPK signaling pathway, and its antioxidative and antiapoptotic effects. In vivo experiments showed that TMP restored PTK787-induced damage to intersegmental vessels (ISVs) in Tg(fli-1: EGFP)y1 transgenic (Flik) zebrafish larvae. Similarly, adrenalin hydrochloride– (AH–) induced reactive oxygen species (ROS) production and thrombosis in AB strain zebrafish were inhibited by TMP. RT-qPCR assay proved that TMP could inhibit the expression of fga, fgb, fgg, f7, and von Willebrand factor (vWF) mRNA to exert an antithrombotic effect. Our findings suggest that TMP can contribute to endothelial injury protection and antithrombosis by modulating MAPK signaling and attenuating oxidative stress and antiapoptosis.