Presentación realizada en el CAECIHS de la UAI (Universidad Abierta Interamericana).

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

2. 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 .

3. “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.”

4. Estadios del desarrollo de zebrafish.

5. 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:10.3791/4196
http://www.jove.com/video/4196/regular-care-maintenance-zebrafish-danio-rerio-laboratory-an
Hembras
(B y C)
Macho
(A)

6. 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.

7. Acuario

8. Manejo de Zebrafish (Danio rerio) en el Laboratorio.
Hembra adulta de 4 meses de edad y 49 mm de longitud.

9. 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.

10. 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).

11. 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.

12. 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/

13. 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.

14. 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.

15. 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

16. 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

17. 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)

18. 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

19. 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.