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ZEBRAFISH: BIOMODELO & BIOINDICADOR María Beatriz Espinosa (Ph.D)

Zebrafish Model & Atherosclerosis

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Page 1: Zebrafish Model & Atherosclerosis

ZEBRAFISH: BIOMODELO & BIOINDICADOR

María Beatriz Espinosa (Ph.D)

Page 2: Zebrafish Model & Atherosclerosis

Danio rerio constituye un modelo

actualmente indiscutible en biomedicina.

Útil para estudiar efectos tóxicos de

sustancias químicas presentes en el agua,

como modelo para aterosclerosis y otras .

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“Many researchers and clinicians agree that the treatment of atherosclerosis must begin at the earliest possible stage – the fatty streak,” said Miller.

“By feeding HCD to zebrafish, we were able to reproduce many of the processes involved in early atherogenesis. Our results suggest that this new model is suitable for studying inflammatory processes that occur in the early development of the disease, by looking at the function of vascular cells and lipid deposits in a live animal.”

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Estadios del desarrollo de zebrafish.

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Cuidado y Mantenimiento de Zebrafish (Danio rerio) en el Laboratorio.

 

Avdesh, A., Chen, M., Martin-Iverson, M. T., Mondal, A., Ong, D., Rainey-Smith, S., … Martins, R. N. (2012). Regular Care and Maintenance of a Zebrafish (Danio rerio) Laboratory: An Introduction. Journal of Visualized Experiments : JoVE, (69), 4196. doi:10.3791/4196http://www.jove.com/video/4196/regular-care-maintenance-zebrafish-danio-rerio-laboratory-an

 

Hembras(B y C)

Macho(A)

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PARÁMETRO RANGO ÓPTIMO

Alcalinidad 50-150 mg/L CaCO3

pH 6.8-7.5 (6.0-8.5 tolerable)

Temperatura 26 - 28.5 °C

Dureza 50-100 mg/L CaCO3

NH4 (Amonio no iónico) <0.02 mg/L

Nitratos (NO3-) <50 mg/L

Nitritos (NO2-) <0.1 mg/L

Oxígeno disuelto >6.0 mg/L

Salinidad 0.5-1 g/L

Conductividad 300 -1,500 μS

Condiciones del agua.

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Acuario

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Manejo de Zebrafish (Danio rerio) en el Laboratorio.

Hembra adulta de 4 meses de edad y 49 mm de longitud.

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Embrión de 24 horas.

-Helenius, I. T., & Yeh, J.-R. J. (2012). Small zebrafish in a big chemical pond.Journal of Cellular Biochemistry, 113(7), 2208–2216. doi:10.1002/jcb.24120

Desarrollo rápido y visualización sencilla de tejidos y órganos durante el desarrollo de los embriones.

Células musculares y estructura de los

miótomos.

Glóbulos rojos y vasos.

Cardiomiocitos (verde)

Estructura del cerebro.

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Simmons, A. E., Karimi, I., Talwar, M., & Simmons, T. W. (2012). Effects of Nitrite on Development of Embryos and Early Larval Stages of the Zebrafish (Danio rerio). Zebrafish, 9(4), 200–206. doi:10.1089/zeb.2012.0746

 A: Sin nitritos (0 mg/L)

Efectos de la exposición a nitritos en larvas de zebrafish.

Vista dorsal de la larva de 5 días (120 horas post

fertilización .

B: Expuesta a nitritos (1500 mg/L  durante 24

horas = hpf).

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Simmons et al., (2012).

A: en 300 mM de etanol durante 24 horas luego de la fertilización.

Vista antero-lateral de la larva de 5 días.

Teratogénesis causada por etanol y nitritos.

B : 300 mg/L de nitritos durante 96 horas.

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Helenius, I. T., & Yeh, J.-R. J. (2012). Small zebrafish in a big chemical pond. Journal of Cellular Biochemistry, 113(7), 2208–2216. doi:10.1002/jcb.24120

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3349782/

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Pancreas, Liver, Gut screens using in situ hybridization.

Fishman, M. C. (1999). Zebrafish genetics: The enigma of arrival. Proceedings of the National Academy of Sciences of the United States of America, 96(19), 10554–10556.

Genética y zebrafish: visualizacion de los órganos embrionarios “in vivo” .

.

Thymus and neural tube after green fluorescent protein transgenesis (GFP= green fluorescent protein).

Heart and Notochord, when enhanced by fluorescence

Blood vessel after injection of fluorescent dextran to fill the vascular tree.

Kidney: Immunohistochemistry.

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Respuesta angiogénica observada por microscopía confocal.

Blood vessels of transgenic fli1a:EGFP zebrafish embryos can be easily observed under the confocal microscope (A), and SIVs are marked by white arrows. Angiogenic responses (B and C) are triggered by tumor cell xenografts and targeted antiangiogenic therapy of RGD-SWCNT(Rh)-thalidomide (D, E and F) in transgenic fli1a:EGFP zebrafish embryos. Engraftment of human HT1080 fibrosarcoma cells, which secrete vascular endothelial growth factors, triggers ectopic angiogenesis of SIVs (B and C). Note morphological features of angiogenic response with engraftment of human HT1080 fibrosarcoma cells. When coinjected with RGD-SWCNT(Rh)-thalidomide (E), ectopic growth of angiogenesis of the SIV is obviously inhibited (D and F) in treated zebrafish embryos. White arrows (E and F) indicate presence of RGD-SWCNT(Rh)-thalidomide after injection. (F) is the merge of (D) and (E). Scale bar: 200 μm.

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Zebrafish & Angiogénesis

Cheng, J., Gu, Y.-J., Wang, Y., Cheng, S. H., & Wong, W.-T. (2011). Nanotherapeutics in angiogenesis: synthesis and in vivo assessment of drug efficacy and biocompatibility in zebrafish embryos. International Journal of Nanomedicine, 6, 2007–2021. doi:10.2147/IJN.S20145

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Stoletov, K., Fang, L., Choi, S.-H., Hartvigsen, K., Hansen, L. F., Hall, C., … Miller, Y. I. (2009). Vascular lipid accumulation, lipoprotein oxidation and macrophage lipid uptake in hypercholesterolemic zebrafish. Circulation

Research, 104(8), 952–960.

“Here we report that feeding adult zebrafish (Danio rerio) a high-cholesterol diet (HCD) resulted in hypercholesterolemia, remarkable lipoprotein oxidation and fatty streak formation in the arteries”.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2834250

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Vascular lipid accumulation, lipoprotein oxidation and macrophage lipid uptake in hypercholesterolemic zebrafish.

E = antibody against L-plastin (macrophages) counterstained with DAPI (nuclei)

A = fatty streaks in the dorsal aorta.

Melanocytes (mln) accumulate around blood vessels. 

DA = Dorsal aorta. CV= Caudal vein; ISA = inter-segmental artery bifurcation. mln, melanocytes.

HCD-fed

van Gieson staining

control

5 μm 20 μm

D and E = DA stained with LipidTOX Red (neutral lipid; merged fluorescent and bright field images)

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Zebrafish y Nicotina

Klee, E. W., Ebbert, J. O., Schneider, H., Hurt, R. D., & Ekker, S. C. (2011). Zebrafish for the Study of the Biological Effects of Nicotine. Nicotine & Tobacco Research, 13(5), 301–312. doi:10.1093/ntr/ntr010 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3145391/

Kedikian X, Faillace MP, Bernabeu R (2013) Behavioral and Molecular Analysis of Nicotine-Conditioned Place Preference in Zebrafish. PLoS ONE 8(7): e69453. doi: 10.1371/journal.pone.0069453

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Nanotherapeutics in

angiogenesis: synthesis and

in vivo assessment of drug

efficacy and biocompatibility

in zebrafish embryos

(Jinping Cheng, et al., 2011).

Eficacia de la biodistribución,

validación en zebrafish.

In vivo biodistribution of Rh-SWCNT-RGD (rhodamine - Single-Walled Carbon Nanotubes - Arginylglycylaspartic acid (RGD) in developing zebrafish embryos at different developmental stages. Zebrafish embryos were loaded with 2 nL of Rh-SWCNT-RGD (2.4 ng of SWCNT and 0.3 ng of RGD) into embryonic cells at the one-cell stage through microinjection.