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ZEBRAFISH: BIOMODELO & BIOINDICADOR
María Beatriz Espinosa (Ph.D)
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 .
“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.”
Estadios del desarrollo de zebrafish.
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)
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.
Acuario
Manejo de Zebrafish (Danio rerio) en el Laboratorio.
Hembra adulta de 4 meses de edad y 49 mm de longitud.
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.
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).
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.
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/
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.
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.
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
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
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)
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
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.