ZEBRAFISH, DANIO RERIO AS A

Transcription

1 ZEBRAFISH, DANIO RERIO AS A REPLACEMENT ALTERNATIVE MANGALA GUNATILAKE Dept. of Physiology Faculty of Medicine University of Colombo

2 Overview: Introduction Objective Methods Concept of Replacement Alternatives and Relative replacement Sharing information on scientific attributes of ZF model and the plan under the Sri Lankan context ZF model in CKDu - with ISO and OECD Acknowledgements

3 1. Reduction Alternatives 2. Refinement Alternatives Russell & Burch in Replacement Alternatives Alternative Any change in an animal test that achieves one or more of the 3Rs

4 As per Russell & Burch, REPLACEMENT alternative technique Any scientific method employing non-sentient material replacing methods which use conscious living vertebrates Invertebrates and Non-mammalian animal models

5 ABSOLUTE RELATIVE Abolitionists Animal researchers

6 Zebrafish, Danio rerio Relative Replacement

7 Fresh water fish Cyprinidae family India Pakistan Nepal Myanmar Bangladesh Most popular vertebrate model organism in ecotoxicology

8 Introduced to Sri Lankan Researchers at the pre & post Conference workshops of the Inaugural Scientific Sessions of the SLALAS : January 2014 Dr Francois Busquet, PhD Center for Alternatives to Animal Testing (CAAT) Europe Policy Coordinator

9 Native fish Ornamental purposes only Unaware of its scientific importance as a research model

10 Wide spectrum of scientific applicability Vertebrate developmental biology s Genetics Neuroscience Physiology Developmental toxicity (teratogenecity) Human disease model Zebrafish & fertilized embryo Toxicology Biomedicine Mammalian organotoxicity

11 Attributes for scientific importance Genetic similarity to humans Small size - Large numbers in a small space Easy maintenance & economic benefits Rapid embryonic development Life span 2 4 years up to 6 years

12 Embryos are permeable to drugs - easy manipulation Development of embryo and larvae Transparent External fertilization High spawning rate

13

14 Copyright: R. A. McKee and R. A. Wingert. Zebrafish Renal Pathology: Emerging Models of Acute Kidney Injury. Curr Pathobiol Rep (2015) 3:

15 Fish Husbandry in a laboratory setting Housing Water quality Purification of water - filters Feeding Health monitoring and diseases Reproduction conditions required

16 Development Stages of Embryos Period hpf Description Zygote 0 Newly fertilized egg Cleavage Beginning of Cleavage cycles Blastula Formation of yolk syncytial layer forms Beginning of Epiboly formation Gastrula Formation of Epiboly Segmentation Somite formation, Organogenensis, Beginning of Movement Pharyngula Beginning of Circulation & Pigmentation, Development of Fins Hatching Completion of primary organs, Hatching begins Early larva 72 Inflation of Swim bladder & further development, Capable in swimming in shallow water Digestive tract development Mid larval Completion of Fins and pigmentation, swim bladder, GIT formation 16

17 somites Head Tail Four-cell-stage approximately 1 hpf Segmentation phase approximately 12 hpf Stages of Embryonic Development of the Zebrafish CHARLES B. KIMMEL, WILLIAM W. BALLARD, SETH R. KIMMEL, BONNIE ULLMANN, AND THOMAS F. SCHILLING. DEVELOPMENTAL DYNAMICS 203: (1995) 17

18 After 24 h Tail is detached from the yolk and spontaneous movement starts After 48 h well structured spine, blood circulation and regular heart beats Dr Francois Busquet. Development of a new screening assay to identify proteratogenic compounds using Zebrafish Danio rerio embryo combined with an exogenous mammalian metabolic activation system (mdart). PhD Thesis. Toxicology institute of Merck KGaA in Darmstadt, Germany,

19 All major organs appear within 36 hpf and hatching takes place h later 19

20 E,F: Protruding-mouth stage (72 h). Progressive increase dorsally in yellow pigmentation owing to xanthophore development, and the progressive filling of melanophores into the lateral stripe. 20 G,H:The early larva (120 h) Inflation of swim bladder

21 Zebrafish (Danio rerio) embryo at 72 hpf Sacculi/ otoliths Chorda/Spinal cord Brain Anus Tail Tail tip Eye Heart Yolk sac & extension

22 Plan in SL ZF in CKDu

23 Clinical Kidney Journal, 2015, 1 7 CKD is uniformly distributed in all provinces while CKDu is restricted to a few provinces.

24 CKDu First patient in 1994 Multifactorial Over 1,100 CKDu patients are hospitalized per month in Sri Lanka and 300 deaths recorded per year > 70,000 patients in SL yrs affected..trend is changing very young Prevalence among farming communities Slowly progressing and irreversible Asymptomatic until late stages Tubulointerstitial fibrosis and tubular atrophy in renal biopsies

25 For water quality and toxicity: International Standardization Organization : ISO 15088:2007(E) Determination of the acute toxicity of waste water to zebrafish eggs (Danio rerio) Organization of Economic Cooperation and Development : OECD TG Fish embryo acute toxicity test (FET) with the ZF for the testing of chemicals No ethics approval needed

26 Scheme of the zebrafish embryo acute toxicity test procedure TG hpf 1.5 hpf 3 hpf Spawning unit Glass vessel with respective test concentrations/co ntrols at volumes to fully cover eggs Selection of fertilized eggs and fertilizing rate Fertilized eggs Removal of coagulated eggs Duration of the test : 96 h

27 Based on the 4 End points, toxicity of substances will be determined. Coagulated embryo Non-detachment of the tail somites No somite formation Lack of heart beat

28 Other tests: Fish Acute Toxicity Test (OECD 203) with whole fish 96 h Fish Early-life Stage Toxicity Test (OECD 210) 30 days post hatch - cumulative mortality - numbers of healthy fish at end of test - time to start of hatching and end of hatching - numbers of larvae hatching each day - length and weight of surviving animals - numbers of deformed larvae - numbers of fish exhibiting abnormal behaviour Fish Juvenile Growth Test (OECD 215) 28 days

29 Plan: 1. Collection of water samples from CKDu endemic and non-endemic areas 2. Performance of tests with Collected water samples Filtered water samples using special filters Suspected heavy metals in different concentrations 3. Histopathology with H & E for renal effects Whole embryo Harvested kidneys of adult fish

30 Dr Hajime Kojima - co-chairman of the Congress and members of the organizing committee Prof M A Akbarsha Director, Mahatma Gandhi Doerenkamp Center for Alternatives to Use of Animals in Life Science Education, India Distinguished Audience