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Immunology and Infection

Oral Intubation of Adult Zebrafish: A Model for Evaluating Intestinal Uptake of Bioactive Compounds

Published: September 27th, 2018

DOI:

10.3791/58366

1Institute of Biotechnology and Biomedicine (IBB-UAB), Universitat Autònoma de Barcelona, 2Department of Cell Biology, Animal Physiology and Immunology, Universitat Autònoma de Barcelona

The protocol describes intubating adult zebrafish with a biologic; then dissecting and preparing the intestine for cytometry, confocal microscopy and qPCR. This method allows administration of bioactive compounds to monitor intestinal uptake and the local immune stimulus evoked. It is relevant for testing the intestinal dynamics of oral prophylactics.

Most pathogens invade organisms through their mucosa. This is particularly true in fish as they are continuously exposed to a microbial-rich water environment. Developing effective methods for oral delivery of immunostimulants or vaccines, which activate the immune system against infectious diseases, is highly desirable. In devising prophylactic tools, good experimental models are needed to test their performance. Here, we show a method for oral intubation of adult zebrafish and a set of procedures to dissect and prepare the intestine for cytometry, confocal microscopy and quantitative polymerase chain reaction (qPCR) analysis. With this protocol, we can precisely administer volumes up to 50 µL to fish weighing approximately 1 g simply and quickly, without harming the animals. This method allows us to explore the direct in vivo uptake of fluorescently labelled compounds by the intestinal mucosa and the immunomodulatory capacity of such biologics at the local site after intubation. By combining downstream methods such as flow cytometry, histology, qPCR and confocal microscopy of the intestinal tissue, we can understand how immunostimulants or vaccines are able to cross the intestinal mucosal barriers, pass through the lamina propria, and reach the muscle, exerting an effect on the intestinal mucosal immune system. The model could be used to test candidate oral prophylactics and delivery systems or the local effect of any orally administered bioactive compound.

The goal of this article is to describe in depth a straightforward method for oral intubation of zebrafish, along with useful associated downstream procedures. Oral intubation using zebrafish has become a practical model in the study of infectious disease dynamics, oral vaccine/immunostimulant, drug/nanoparticle uptake and efficacy, and intestinal mucosal immunity. For example, zebrafish oral intubation has been used in the study of Mycobacterium marinum and Mycobacterium peregrinum infection1. Lovmo et al. also successfully used this model to deliver nanoparticles and M. marinum to the gastro-intestinal trac....

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All experimental procedures involving zebrafish (Danio rerio) were authorized by the Ethics Committee of the Universitat Autònoma de Barcelona (CEEH number 1582) in agreement with the International Guiding Principles for Research Involving Animals (EU 2010/63). All experiments with live zebrafish were performed at 26–28 °C.

1. Preparing the Equipment for Oral Intubation

  1. Place approximately 1 cm of a fine silicone tube on a 31 G Luer lock needle to cover the.......

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Zebrafish (average weight: 1.03 ± 0.16 g) of mixed sex were successfully intubated with different recombinant protein nanoparticles (bacterial inclusion bodies) using our home-made oral intubation device (Figure 1). We have successfully performed the oral intubation and achieved a low average percentage mortality (6.8%) (Table 1). Zebrafish were either intubated with 30 µL or 50 µL of nanoparticle suspensions and the mortality .......

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This protocol is an improvement of the previously described technique for oral intubation by Collymore et al.4 Our protocol describes in detail the oral intubation method and includes the preparation of the intestine for downstream analyses. Our method improves fish manipulation speed allowing one person to perform the whole protocol rapidly, without much variation between operators. A main difference of our protocol with the previous one is that we evaluate the success of an oral intubat.......

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This work was supported by grants from the Spanish Ministry of Science, European commission and AGAUR funds to NR (AGL2015-65129-R MINECO/FEDER and 2014SGR-345 AGAUR). RT holds a pre-doctoral scholarship from AGAUR (Spain), JJ was supported by a PhD fellowship from the China Scholarship Council (China) and NR is supported by the Ramón y Cajal program (RYC-2010-06210, 2010, MINECO). We thank Dr. Torrealba for expert advice in protein production, N. Barba from the "Servei de Microscopia" and Dr. M. Costa from the "Servei de Citometria" of the Universitat Autònoma de Barcelona for helpful technical assistance.

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Name Company Catalog Number Comments
Silicon tube Dow Corning 508-001 0.30 mm inner diameter and 0.64 mm outer diameter
Luer lock needle Hamilton 7750-22 31 G, Kel-F Hub
Luer lock syringe Hamilton 81020/01 100 μL, Kel-F Hub
Filtered pipette tip Nerbe Plus 07-613-8300 10 μL
MS-222 Sigma Aldrich E10521 powder
10x PBS Sigma Aldrich P5493
Filter paper  Filter-Lab RM14034252
Collagenase Gibco 17104019
DMEM  Gibco 31966 Dulbecco's modified eagle medium
Penicillin and streptomycin Gibco 15240
Cell strainer Falcon 352360
CellTrics filters  Sysmex Partec 04-004-2326 (Wolflabs) 30 µm mesh size filters with 2 mL reservoir
Tissue-Tek O.C.T. compound SAKURA 4583
Plastic molds for cryosections SAKURA 4557 Disposable Vinyl molds. 25 mm x 20 mm x 5 mm
Slide Thermo Scientific 10149870 SuperFrost Plus slide
Cover glasses Labbox  COVN-024-200 24´24 mm
Paraformaldehyde (PFA) Sigma-Aldrich 158127
Atto-488 NHS ester Sigma-Aldrich 41698
Sodium bicarbonate Sigma-Aldrich S5761
DMSO Sigma-Aldrich D8418
Maxwell RSC simplyRNA Tissue Kit Promega AS1340
1-Thioglycerol/Homogenization solution Promega Inside of Maxwell RSC simplyRNA Tissue Kit adding 20 μl 1-Thioglycerol to 1 ml homogenization solution (2%)
vertical laboratory rotator  Suministros Grupo Esper 10000-01062
Cryostat Leica  CM3050S
Homogenizer KINEMATICA Polytron PT1600E
Flow cytometer  Becton Dickinson FACS Canto
5 mL round bottom tube Falcon 352058
Confocal microscope Leica SP5
Fume Hood Kottermann 2-447 BST
Nanodrop 1000 Thermo Fisher Scientific ND-1000 Spectrophotometer
Agilent 2100 Bioanalyzer System Agilent G2939A RNA bioanalyzer
Maxwell Instrument Promega AS4500 
iScript cDNA synthesis kit  Bio-rad 1708891
CFX384 Real-Time PCR Detection System Bio-Rad 1855485
iTaq universal SYBR Green Supermix kit Bio-rad 172-5120
Water  Sigma-Aldrich W4502
Cryogenic vial  Thermo Fisher Scientific 375418 CryoTube vial
Mounting medium Sigma-Aldrich F6057 Fluoroshield with DAPI

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