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1.
Micron ; 130: 102801, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31864139

RESUMO

Over the past few decades, Zebrafish has become a widely used vertebrate model for cardiovascular research. Easy genetic manipulation, low cost, high fecundity, embryonic transparency, and ability to survive in the early stages of development without active circulation are among the advantages of Zebrafish. Cardiac malformations can be induced through genetic manipulations for elucidating the influence of mechanobiological stimuli on the development and progress of the cardiovascular diseases. For this purpose, a reliable in vivo assessment of cardiac function and disturbed hemodynamics is required. Therefore, it is necessary to accurately determine the complex blood flow patterns and associated hemodynamic shear stresses within the developing heart and cardiovascular system. In the traditional approach, brightfield microscopy is used to track the motion of cells in two-dimensions (2D). However, with the development of advanced modalities such as light-sheet fluorescent microscopy, it is now possible to perform 4D (three-dimensional space + time) imaging of Zebrafish embryo and larvae. The integration of digital particle image velocimetry (DPIV) and computational fluid dynamics (CFD) provide an opportunity for detailed investigations using in vivo images. In this review, DPIV and CFD methods are explained for blood flow assessment, and recent relevant research findings from Zebrafish studies are summarized.


Assuntos
Simulação por Computador , Hemodinâmica , Reologia/métodos , Peixe-Zebra/fisiologia , Animais , Hidrodinâmica , Estresse Mecânico , Peixe-Zebra/sangue
2.
J Am Assoc Lab Anim Sci ; 58(6): 823-828, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31662156

RESUMO

Zebrafish are an important model in neuroscience and developmental biology and are also an emerging model in hematology and immunology. Little information is available for zebrafish regarding the physiologic impact of different euthanasia methods and whether a chosen method of euthanasia can impact serum yield. These parameters could impact the choice of euthanasia method for a study. To that end, the current study compared 3 methods of adult zebrafish euthanasia and their effects on 3 distinct criteria; time to loss of opercular movement, volume of serum obtained, and serum cortisol concentration. Blood was collected using a postmortem tail amputation and centrifugation blood collection technique. Time to loss of opercular movement differed significantly among euthanasia methods, with animals undergoing rapid chilling displaying the shortest time (mean Rapid Chilling: 40 s; Benzocaine: 86 s; MS222: 96 s). All methods of euthanasia resulted in a comparable average serum yield (Rapid Chilling = 7.5 µL; Benzocaine = 8.5 µL; MS222 = 7.5 µL per fish). None of the euthanasia methods tested resulted in average cortisol concentrations above the reported physiologic range. Although no significant differences were observed in serum yield or serum cortisol concentration, rapid chilling remains the preferred method for painless, humane euthanasia.


Assuntos
Anestésicos/farmacologia , Benzocaína/farmacologia , Eutanásia Animal/métodos , Hidrocortisona/sangue , Peixe-Zebra/sangue , Animais , Humanos
3.
Aquat Toxicol ; 216: 105290, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31518775

RESUMO

Phthalate esters (PAEs), which are notable plasticizers, can be prolific contaminants in aquatic environments, and have been shown to induce reproductive toxicity. However, the studies concerning their toxicity towards aquatic species are based on individual chemicals, and the combined toxicity of PAEs to aquatic organisms remains unclear. The aim of this study was to explore the potential toxicity mechanisms associated with combined exposure to dibutyl phthalate (DBP) and diisobutyl phthalate (DiBP) in adult female zebrafish ovaries. Zebrafish were exposed to DBP, DiBP and their mixtures for 30 days, and their effects on ovarian histology, plasma sex hormones and ovarian transcriptomics were investigated. Plasma estradiol (E2) levels were significantly decreased by 38.9% in the DBP-1133 exposure group and 41.0% in the DiBP-1038 exposure group. The percentage of late/mature oocytes was also significantly decreased by 17.3% under DBP-1133 exposure and 16.2% under DiBP-1038 exposure, while that under combined exposure was not significantly affected. Nevertheless, transcriptome sequencing revealed 2564 differentially expressed genes (DEGs) in zebrafish ovaries after exposure to the mixtures. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the DEGs were involved in the neuroactive ligand-receptor interaction, GnRH, progesterone-mediated oocyte maturation, oocyte meiosis and steroid hormone biosynthesis signaling pathways. These results revealed that combined exposure exerts potential reproductive toxicity at the molecular level.


Assuntos
Dibutilftalato/análogos & derivados , Dibutilftalato/toxicidade , Exposição Ambiental , Perfilação da Expressão Gênica , Ovário/metabolismo , Transcriptoma/genética , Peixe-Zebra/genética , Animais , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Ontologia Genética , Hormônios Esteroides Gonadais/sangue , Ovário/efeitos dos fármacos , Ovário/patologia , Reprodutibilidade dos Testes , Reprodução/efeitos dos fármacos , Reprodução/genética , Poluentes Químicos da Água/toxicidade , Peixe-Zebra/sangue
4.
Development ; 146(16)2019 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-31375478

RESUMO

How developing vascular networks acquire the right balance of arteries, veins and lymphatic vessels to efficiently supply and drain tissues is poorly understood. In zebrafish embryos, the robust and regular 50:50 global balance of intersegmental veins and arteries that form along the trunk prompts the intriguing question of how does the organism keep 'count'? Previous studies have suggested that the ultimate fate of an intersegmental vessel (ISV) is determined by the identity of the approaching secondary sprout emerging from the posterior cardinal vein. Here, we show that the formation of a balanced trunk vasculature involves an early heterogeneity in endothelial cell behaviour and Notch signalling activity in the seemingly identical primary ISVs that is independent of secondary sprouting and flow. We show that Notch signalling mediates the local patterning of ISVs, and an adaptive flow-mediated mechanism subsequently fine-tunes the global balance of arteries and veins along the trunk. We propose that this dual mechanism provides the adaptability required to establish a balanced network of arteries, veins and lymphatic vessels.


Assuntos
Padronização Corporal , Receptores Notch/metabolismo , Peixe-Zebra/embriologia , Animais , Artérias/embriologia , Polaridade Celular , Células Endoteliais/fisiologia , Heterogeneidade Genética , Vasos Linfáticos/embriologia , Fluxo Sanguíneo Regional , Transdução de Sinais , Veias/embriologia , Peixe-Zebra/sangue
5.
Aquat Toxicol ; 214: 105240, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31319295

RESUMO

Fish has a strong resistance to microcystins (MCs), cyclic heptapeptide cyanotoxins, known as endocrine disrupting chemicals (EDCs) which are released during cyanobacterial blooms and many laboratory and field studies have found the hepatic recovery of fish from the MCs exposure. The aim of the present study was to investigate the recovery mechanisms of reproductive function of adult zebrafish (Danio rerio) from microcystin-LR (MC-LR) exposure. Therefore, adult female zebrafish were exposed to 0, 1 or 50 µg/L of MC-LR for 21days and transferred to MC free water for another 21 days to investigate the recovery. After MC-LR exposure, marked histological lesions in the gonads, decreased the percentage of mature oocytes, decreased number of spawned eggs, decreased fertilization and hatching rates were observed. MC-LR exposure increased the concentration of 17ß-estradiol (E2), testosterone (T) and vitellogenin (VTG) in female zebrafish. Some gene transcriptions of the hypothalamic-pituitary-gonad (HPG) axis significantly changed. The protein levels of 17ßhsd and cyp19a remarkably increased in the MC-LR exposure groups. However, our laboratory observation also indicates that zebrafish transferred from microcystin exposure to toxin-free water and reared for 21 days exhibited a nearly complete recovery of reproductive functions, including histological structure, increased the percentage of matured oocytes and spawned eggs, stable hormone levels, well-balanced transcriptional and translational levels. These results indicate that after MC-LR exposure, the reproductive impairments in zebrafish are also reversible likewise hepatic recovery seen by different studies in fish. Future studies should be conducted to explore a better understanding of the recovery mechanisms of fish from microcystins exposure.


Assuntos
Exposição Ambiental , Microcistinas/toxicidade , Reprodução/efeitos dos fármacos , Peixe-Zebra/fisiologia , Animais , Disruptores Endócrinos/toxicidade , Feminino , Hormônios/sangue , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Toxinas Marinhas , Ovário/citologia , Ovário/efeitos dos fármacos , Ovário/fisiologia , Hipófise/efeitos dos fármacos , Hipófise/metabolismo , Transcrição Genética/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Peixe-Zebra/sangue , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
6.
Zebrafish ; 16(6): 497-504, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31355732

RESUMO

Palmitic acid (PA) is the most abundant saturated fatty acid in fast foods and is known to induce inflammation and cellular injury in various tissues. In this study, we investigated whether a PA-enriched diet can induce hepatic steatosis and injury in adult zebrafish. The adult zebrafish exhibited increased body weight, hyperlipidemia, hyperglycemia, and steatosis and a hepatic injury phenotype after being fed with a PA-enriched diet for 6 weeks. The quantitative polymerase chain reaction analysis demonstrated that genes associated with hepatic injury were all significantly increased in the liver. Furthermore, livers from the PA-fed group showed an increased messenger RNA (mRNA) expression associated with oxidative stress and endoplasmic reticulum (ER) stress responses. We also found significant upregulation of genes involved in lipid metabolism and triacylglyceride accumulation. Ultrastructural analysis revealed mitochondrial cristae injury and a dilated ER phenotype in the PA-fed hepatocytes, which can be causes of hepatic injury. PA-enriched diet induced hepatic steatosis and injury in adult zebrafish that recapitulated typical metabolic changes and pathophysiological changes as well as increased oxidative stress and ER stress observed in patients with nonalcoholic fatty liver disease.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/patologia , Dieta/efeitos adversos , Fígado Gorduroso/patologia , Ácido Palmítico/efeitos adversos , Peixe-Zebra , Ração Animal/efeitos adversos , Animais , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Fígado Gorduroso/induzido quimicamente , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Masculino , Estresse Oxidativo/efeitos dos fármacos , RNA Mensageiro/análise , Reação em Cadeia da Polimerase em Tempo Real , Peixe-Zebra/sangue
7.
Ecotoxicol Environ Saf ; 182: 109376, 2019 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-31254851

RESUMO

Phenanthrene (PHE) is a tricyclic polycyclic aromatic hydrocarbon which distributed extensively in the aquatic environment. However, the knowledge about its impact on fish reproduction is still limited, particularly under a chronic exposure regime. In this study, we exposed zebrafish (Danio rerio) embryos to environmentally relevant concentrations (0.2, 1.0, and 5.0 µg/L) of PHE for 4 months and assessed the impact on reproduction. The results demonstrated that egg production was decreased in fish exposed to PHE, with a significant reduction at 5.0 µg/L. The exposure significantly decreased the circulating concentrations of estradiol (E2) and testosterone (T) in female fish or E2 in male fish. In addition, plasma vitellogenin levels were significantly inhibited after PHE exposure in female fish. The transcription of hypothalamic-pituitary-gonadal (HPG) axis related genes (GnRH2, FSHß, LHß, 17ß-HSD, CYP11A1, and CYP19a) were significantly altered in a sex-specific manner. In addition, embryos derived from exposed parents exhibited increased malformation and decreased hatching success in the F1 generation. Taken together, these results demonstrate that chronic exposure to environmentally relevant concentration of PHE could cause adverse effects on reproduction and impair the development of offspring, ultimately leading to fish population decline in aquatic environment.


Assuntos
Embrião não Mamífero/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Fenantrenos/toxicidade , Poluentes Químicos da Água/toxicidade , Peixe-Zebra/crescimento & desenvolvimento , Animais , Estradiol/sangue , Feminino , Hormônio Liberador de Gonadotropina/sangue , Masculino , Reprodução/efeitos dos fármacos , Fatores Sexuais , Testosterona/sangue , Vitelogeninas/sangue , Peixe-Zebra/sangue , Peixe-Zebra/genética
8.
Artigo em Inglês | MEDLINE | ID: mdl-31075501

RESUMO

Zebrafish (Danio rerio) are widely used animal models. Nevertheless, the mechanisms underlying hypoxia tolerance in this species have remained poorly understood. In the present study, we have determined the effects of hypoxia on blood-O2 transport properties and mitochondrial respiration rate in permeabilized muscle fibres of adult zebrafish exposed to either 1) a gradual decrease in O2 levels until fish lost equilibrium (~1 h, acute hypoxia), or 2) severe hypoxia (PO2 ∼ 15 Torr) for 48 h (prolonged hypoxia). Acute, short-term hypoxia caused an increase in hemoglobin (Hb) O2 affinity (decrease in P50), due to a decrease in erythrocyte ATP after erythrocyte swelling. No changes in isoHb expression patterns were observed between hypoxic and normoxic treatments. Prolonged hypoxia elicited additional reponses on O2 consumption: lactate accumulated in the blood, indicating that zebrafish relied on glycolysis for ATP production, and mitochondrial respiration of skeletal muscle was overall significantly inhibited. In addition, male zebrafish had higher hypoxia tolerance (measured as time to loss of equilibrium) than females. The present study contributes to our understanding of the adaptive mechanisms that allow zebrafish, and by inference other fish species, to cope with low O2 levels.


Assuntos
Hipóxia/sangue , Músculo Esquelético/metabolismo , Consumo de Oxigênio , Oxigênio/sangue , Peixe-Zebra/sangue , Animais , Transporte Biológico Ativo , Hemoglobinas/metabolismo , Proteínas de Peixe-Zebra/metabolismo
9.
Aquat Toxicol ; 212: 70-76, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31077968

RESUMO

Medroxyprogesterone acetate (MPA) is a widely used synthetic progestin and it has been frequently detected in aquatic environments. However, its effects on aquatic organisms remain largely unknown. Here we investigated the chronic effects of MPA on sex differentiation and gonad development in zebrafish. Zebrafish larvae at 20 days post fertilization (dpf) were exposed to 4.32, 42.0, and 424 ng L-1 of MPA until they reached 140 dpf. The results showed that chronic exposure to 42.0 ng L-1 of MPA caused 60% proportion of males as well as significant up-regulation of dmrt1 (˜1.79 fold) and hsd17b3 (˜1.92 fold). Histological analysis showed MPA significantly increased the frequency of immature spermatocytes accompanied with the increased transcription of dmrt1 (˜2.06 fold) and ar (˜1.73 fold) in the testes. Meanwhile, MPA exposure significantly increased the transcription of lhb at all exposure concentrations in the males. In contrast, it significantly suppressed the transcription of lhb (˜-8.06-fold) and fshb (˜-6.35-fold) at 42.0 ng L-1 in the females. Collectively our results demonstrated that MPA had androgenic activity, and could affect sex differentiation and spermatogenesis in zebrafish at environmentally relevant concentrations. The findings from this study suggest that MPA in the aquatic environment may pose potential androgenic risks to fish populations.


Assuntos
Acetato de Medroxiprogesterona/farmacologia , Diferenciação Sexual/efeitos dos fármacos , Espermatogênese/efeitos dos fármacos , Peixe-Zebra/fisiologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Gônadas/efeitos dos fármacos , Hormônios/sangue , Masculino , Hipófise/efeitos dos fármacos , Hipófise/metabolismo , Razão de Masculinidade , Transcrição Genética/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Peixe-Zebra/sangue , Peixe-Zebra/crescimento & desenvolvimento
10.
Sci Rep ; 9(1): 7131, 2019 05 09.
Artigo em Inglês | MEDLINE | ID: mdl-31073223

RESUMO

The Reprimo gene family comprises a group of single-exon genes for which their physiological function remains poorly understood. Heretofore, mammalian Reprimo (RPRM) has been described as a putative p53-dependent tumor suppressor gene that functions at the G2/M cell cycle checkpoint. Another family member, Reprimo-like (RPRML), has not yet an established role in physiology or pathology. Importantly, RPRML expression pattern is conserved between zebrafish and human species. Here, using CRISPR-Cas9 and antisense morpholino oligonucleotides, we disrupt the expression of rprml in zebrafish and demonstrate that its loss leads to impaired definitive hematopoiesis. The formation of hemangioblasts and the primitive wave of hematopoiesis occur normally in absence of rprml. Later in development there is a significant reduction in erythroid-myeloid precursors (EMP) at the posterior blood island (PBI) and a significant decline of definitive hematopoietic stem/progenitor cells (HSPCs). Furthermore, loss of rprml also increases the activity of caspase-3 in endothelial cells within the caudal hematopoietic tissue (CHT), the first perivascular niche where HSPCs reside during zebrafish embryonic development. Herein, we report an essential role for rprml during hematovascular development in zebrafish embryos, specifically during the definitive waves of hematopoiesis, indicating for the first time a physiological role for the rprml gene.


Assuntos
Hemangioblastos/metabolismo , Proteínas de Membrana/genética , Peixe-Zebra/embriologia , Animais , Sistemas CRISPR-Cas , Proteínas de Ciclo Celular/genética , Desenvolvimento Embrionário , Hematopoese , Morfolinos/farmacologia , Família Multigênica , Peixe-Zebra/sangue , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
11.
Molecules ; 24(8)2019 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-30999617

RESUMO

A standard protocol to develop type 1 diabetes in zebrafish is still uncertain due to unpredictable factors. In this study, an optimized protocol was developed and used to evaluate the anti-diabetic activity of Psychotria malayana leaf. The aims of this study were to develop a type 1 diabetic adult zebrafish model and to evaluate the anti-diabetic activity of the plant extract on the developed model. The ability of streptozotocin and alloxan at a different dose to elevate the blood glucose levels in zebrafish was evaluated. While the anti-diabetic activity of P. malayana aqueous extract was evaluated through analysis of blood glucose and LC-MS analysis fingerprinting. The results indicated that a single intraperitoneal injection of 300 mg/kg alloxan was the optimal dose to elevate the fasting blood glucose in zebrafish. Furthermore, the plant extract at 1, 2, and 3 g/kg significantly reduced blood glucose levels in the diabetic zebrafish. In addition, LC-MS-based fingerprinting indicated that 3 g/kg plant extract more effective than other doses. Phytosterols, sugar alcohols, sugar acid, free fatty acids, cyclitols, phenolics, and alkaloid were detected in the extract using GC-MS. In conclusion, P. malayana leaf aqueous extract showed anti-diabetic activity on the developed type 1 diabetic zebrafish model.


Assuntos
Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/tratamento farmacológico , Hipoglicemiantes/farmacologia , Extratos Vegetais/farmacologia , Psychotria/química , Peixe-Zebra/sangue , Animais , Hipoglicemiantes/química , Extratos Vegetais/química , Extratos Vegetais/farmacocinética
12.
J Am Assoc Lab Anim Sci ; 58(3): 390-396, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30925946

RESUMO

MS222 is a compound used in anesthetizing vertebrates, including fish and frogs. Several side effects of this anesthetic have been reported, but its effect on hemostasis has not been studied. In our laboratory, we have used zebrafish for more than 2 decades as a model system to study hemostasis. During this period, we have had trouble in collecting blood from anesthetized zebrafish and observed more rapid blood clotting than in nonanesthetized counterparts. However, no systematic studies regarding the effect of MS222 on zebrafish hemostasis are available. In this study, we performed various assays such as gill bleeding, measurement of Hct, total blood cell counts, thrombocyte counts, thrombocyte aggregation, and coagula- tion and measured the amount of blood collected. We found that Hct values, the amount of blood collected, bleeding, and coagulation differed significantly between anesthetized and nonanesthetized fish. Our results suggest that blood collected after MS222 anesthesia of zebrafish has altered hemostasis.


Assuntos
Aminobenzoatos/farmacologia , Anestésicos/farmacologia , Hemostasia/efeitos dos fármacos , Peixe-Zebra/sangue , Anestesia , Animais , Contagem de Células Sanguíneas/veterinária , Coagulação Sanguínea/efeitos dos fármacos
13.
Anal Chem ; 91(4): 2744-2751, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30694652

RESUMO

Sialic acid is a family of N- and O-substitutions of neuraminic acid. Plasma or serum sialic acid has been established as a potential disease marker. For example, the presence of 9- O-acetyl on the sialic acid of some glycans and glycoconjugates (e.g., 9- O-acetyl GD3 ganglioside) could be related to cancer occurrence. A variety of assays are available to measure serum or plasma sialic acid; however, sample preparation and storage can alter the O-acetylation profile due to the loss of O-acetyl groups and/or the migration of O-acetyl groups. Herein, we report dried blood spot (DBS) sampling, in combination with diamino-4,5-methylenedioxybenzene derivatization, for profiling sialic acids in blood samples with minimal alteration in O-acetylation patterns. The feasibility of the method was first evaluated by analyzing sialic acids in crucian carp blood and comparing with traditional blood/plasma sample preparation procedures. A total of 19 different sialic acids were identified by using liquid chromatography-Orbitrap mass spectrometry, including four mono-O-acetylated N-acetylneuraminic acids, four mono-O-acetylated N-glycolylneuraminic acids, six di-O-acetylated N-acetylneuraminic acids, and three tri-O-acetylated N-acetylneuraminic acids. The long-term storage study indicated that DBS sampling could effectively preserve the O-acetylation information for at least 6 weeks. Thus, it is demonstrated that this method is a valuable tool for the study of sialic acid diversity, especially for the characterization of isomeric structures.


Assuntos
Teste em Amostras de Sangue Seco , Ácido N-Acetilneuramínico/sangue , Acetilação , Animais , Carpas/sangue , Cromatografia Líquida de Alta Pressão/métodos , Teste em Amostras de Sangue Seco/métodos , Humanos , Espectrometria de Massas/métodos , Ácido N-Acetilneuramínico/química , Peixe-Zebra/sangue
14.
Fish Shellfish Immunol ; 86: 892-899, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30580041

RESUMO

To better understand spring viremia of carp virus (SVCV) pathogenesis in zebrafish proteomic analysis was used to examine the plasma protein profile in SVCV-infected zebrafish. A total of 3062 proteins were identified. Of those 137, 63 and 31 proteins were enriched in blood samples harvested at 1, 2 and 5 days post SVCV infection, respectively. These altered host proteins were classified based on their biological function: 23 proteins under the response to stimulus term were identified. Interestingly, at the top of the up-regulated proteins during SVCV infection were the proteins of the vitellogenin family (Vtg) and the grass carp reovirus-induced gene (Gig) proteins. Real-time RT-PCR evaluation of samples from internal organs verified that SVCV infection induced vtg and gig2 gene expression already at day 1 post-infection. Western blot analysis revealed the presence of Vtg protein only in blood of SVCV-infected fish. This is the first proteomic study to reveal the involvement of Vtg proteins in adult fish response to viral challenge. It also highlights the role of Gig proteins as important factors in antiviral response in fish. This work provides valuable relevant insight into virus-host interaction and the identification of molecular markers of fish response to virus.


Assuntos
Proteínas de Peixes/imunologia , Plasma/química , Proteoma/imunologia , Peixe-Zebra/imunologia , Animais , Doenças dos Peixes/imunologia , Rhabdoviridae/fisiologia , Infecções por Rhabdoviridae/imunologia , Infecções por Rhabdoviridae/veterinária , Peixe-Zebra/sangue , Peixe-Zebra/metabolismo
15.
Microsc Res Tech ; 81(12): 1361-1365, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30431202

RESUMO

A new blood flow imaging (BFI) technique using digital holography with double illumination of the sample is proposed. We imaged the moving red blood cells (RBCs) using a two microscope objective lenses setup. The setup consists in a larger angle of separation (90 °) between the two illumination beams, allowing a wider angular rotation at good z resolution. Moreover, the setup geometry allows an easier displacement of the sample in all directions. Results show that this technique is able to perform phase-shifting reconstruction for the two beams at the same time which is more suitable for the future implementation of live 3D holography. Experimental results are carried out for the verification of the effectiveness of the proposed technique on a zebrafish larvae sample. RESEARCH HIGHLIGHTS: Blood flow imaging techniques are often invasive and image analysis is time consuming. To alleviate this issue an imaging technique based on dual illumination in holographic domain is proposed. This method has been validated on zebrafish embryos.


Assuntos
Holografia/métodos , Imageamento Tridimensional/métodos , Microscopia/métodos , Peixe-Zebra/sangue , Animais , Desenho de Equipamento , Eritrócitos/química , Eritrócitos/citologia , Larva/química , Larva/fisiologia , Peixe-Zebra/embriologia , Peixe-Zebra/fisiologia
16.
Methods Mol Biol ; 1797: 487-495, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29896711

RESUMO

Investigations of teratogenic effects of drugs generally involve testing the drug on animals and zebrafish embryo is a commonly used animal model for that purpose. In these studies, cardiovascular function of the animals needs to be evaluated to reveal the influence of exposure on the development of the cardiovascular system as well as on the growth of the whole animal. Here, relevant microscopy imaging and analysis protocols are described to calculate a variety of hemodynamic parameters for zebrafish embryos exposed to clinical drugs.


Assuntos
Embrião não Mamífero/irrigação sanguínea , Embrião não Mamífero/efeitos dos fármacos , Hemodinâmica , Teratogênese/efeitos dos fármacos , Teratogênios/farmacologia , Peixe-Zebra/sangue , Animais , Fenômenos Fisiológicos Cardiovasculares , Sistema Cardiovascular/embriologia , Imagem Molecular/métodos , Peixe-Zebra/embriologia
17.
Aquat Toxicol ; 200: 206-216, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29775928

RESUMO

Tributyltin (TBT), an organotin acting as aromatase (Cyp19a1) inhibitor, has been found to disrupt gametogenesis and reproductive behaviors in several fish species. However, few studies addressing the mechanisms underlying the impaired gametogenesis and reproduction have been reported. In this study, female adults of zebrafish (Danio rerio) were continuously exposed to two nominal concentrations of TBT (100 and 500 ng/L, actual concentrations: 90.8 ±â€¯1.3 ng/L and 470.3 ±â€¯2.7 ng/L, respectively) for 28 days. After exposures, TBT decreased the total egg number, reduced the hatchability and elevated the mortality of the larvae. Decreased gonadosomatic index (GSI) and altered percentages of follicles in different developmental stages (increased early-stage follicles and reduced mid/late-stage follicles) were also observed in the ovary of TBT-treated fish. TBT also lowered the plasma level of 17ß-estradiol and suppressed the expressions of cyp19a1a in the ovary. In treated fish, up-regulated expressions of aldhla2, sycp3 and dmc1 were present in the ovary, indicating an enhanced level of meiosis. The mRNA level of vtg1 was dramatically suppressed in the liver of TBT-treated fish, suggesting an insufficient synthesis of Vtg protein, consistent with the decreased percentage of mid/late-stage follicles in the ovaries. Moreover, TBT significantly suppressed the reproductive behaviors of the female fish (duration of both sexes simultaneously in spawning area, the frequency of meeting and the visit in spawning area) and down-regulated the mRNA levels of genes involved in the regulation of reproductive behaviors (cyp19a1b, gnrh-3 and kiss 2) in the brain. In addition, TBT significantly suppressed the expressions of serotonin-related genes, such as tph2 (encoding serotonin synthase), pet1 (marker of serotonin neuron) and kiss 1 (the modulator of serotonin synthesis), suggesting that TBT might disrupt the non-reproductive behaviors of zebrafish. The present study demonstrated that TBT may impair the reproductive success of zebrafish females probably through disrupting oogenesis, disturbing reproductive behaviors and altering serotonin synthesis. The present study greatly extends our understanding on the reproductive toxicity of TBT on fish.


Assuntos
Oogênese/efeitos dos fármacos , Reprodução/efeitos dos fármacos , Serotonina/biossíntese , Compostos de Trialquitina/toxicidade , Peixe-Zebra/fisiologia , Animais , Comportamento Animal/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Estradiol/sangue , Feminino , Larva/efeitos dos fármacos , Larva/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Meiose/efeitos dos fármacos , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Serotonina/metabolismo , Vitelogeninas/genética , Vitelogeninas/metabolismo , Poluentes Químicos da Água/toxicidade , Peixe-Zebra/sangue
18.
Vet Immunol Immunopathol ; 199: 39-42, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29678228

RESUMO

Alternative complement activity was determined in whole body homogenates (WBHs) and serum samples of different fish species, by measuring the amount of sample that induces 50% hemolysis of red blood cells using the ACH50 assay (Alternative Complement pathway Hemolytic activity). Values of ACH50 obtained for serum samples were about two-fold higher when using rabbit red blood cells (RRBC), as compared to sheep red blood cells (SRBC). The increase in ACH50 when using RRBCs for WBH samples was 28, 7 and 4 folds for guppy, molly and zebrafish, respectively. Large variability in complement activity was evident between fish species for both serum and WBHs. Evaluating the effect of freeze-thaw cycles on complement revealed significant reduction in complement activity in all tested samples. Loss of activity following three freeze-thaw cycles amounted to 48-59% when serum was tested and over 95% loss in activity for WBH. To our knowledge, this is the first study where fish WBHs were used for assaying complement activity. Our results support the suitability of this method in evaluating complement activity in small fish species or larvae, where blood cannot be obtained, as long as samples can be tested upon first thawing.


Assuntos
Ativação do Complemento/imunologia , Eritrócitos/imunologia , Peixes/imunologia , Animais , Via Alternativa do Complemento/imunologia , Peixes/sangue , Hemólise , Poecilia/sangue , Poecilia/imunologia , Coelhos/sangue , Ovinos/sangue , Peixe-Zebra/sangue , Peixe-Zebra/imunologia
19.
Microsc Res Tech ; 81(2): 153-161, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27155205

RESUMO

Microvessel blood flow imaging techniques are widely used in biomedical research and clinical diagnostics where many diseases have a vascular etiology or involvement. For testing purposes, zebrafish embryo provides an ideal animal model to achieve high-resolution imaging of superficial and deeply localized vessels. Moreover, the study of the formation of a closed circulatory system in vertebrates is a topic of recent interest in biophysics. However, most of the existing techniques are invasive due to the use of a contrast agent for imaging purposes. Recent developments in Digital Holography and Laser Doppler Holography techniques can be considered to alleviate this issue. Laser Doppler holography and transmission microscopy can be coupled to analyze blood flow in fish embryos by adapting a laser Doppler holographic setup to a standard bio-microscope: the two beams of the holographic interferometer (illumination of the object and reference), whose frequency offset is controlled, were addressed to the microscope by optical fibers. Multimodal acquisition and analysis of the data is made by acting on the frequency offset of the two beams, and on the location of the Fourier space filtered zone. In this work, we show that it is possible to select the signal of moving scatterers, and to image Red Blood Cells (RBCs) and blood vessels. Individual RBCs are imaged, and movies showing the RBC motion are obtained. Microsc. Res. Tech. 81:153-161, 2018. © 2016 Wiley Periodicals, Inc.


Assuntos
Holografia/métodos , Microvasos/diagnóstico por imagem , Peixe-Zebra/anatomia & histologia , Animais , Fenômenos Fisiológicos Sanguíneos , Fluxometria por Laser-Doppler/métodos , Peixe-Zebra/sangue , Peixe-Zebra/embriologia
20.
J Biophotonics ; 11(2)2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-28700144

RESUMO

We present an in vivo lab-free full-field functional optical hemocytometer (FFOH) for application to the capillaries of a live biological specimen, based on the absorption intensity fluctuation modulation (AIFM) effect. Because of the absorption difference between the red blood cells (RBCs) and background tissue under low-coherence light illumination, an endogenous instantaneous intensity fluctuation is generated by the AIFM effect when RBCs discontinuously traverse the capillary. The AIFM effect is used to highlight the RBC signal relative to the background tissue by computing the real-time modulation depth. FFOH can simultaneously provide a flow video, the flow velocity and the RBC count. Ourexperimental results can potentially be applied to study the physiological mechanisms of the blood circulation systems of near-transparent live biological samples.


Assuntos
Contagem de Eritrócitos/instrumentação , Dispositivos Ópticos , Animais , Circulação Sanguínea , Capilares/fisiologia , Peixe-Zebra/sangue
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