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

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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 needle tip.
2. Cut a 10 µL sterile filter pipette tip (around 2 cm), take the finer end and place it over the silicone tube as a sheath. ....

Results

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 .......

Discussion

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.......

Materials

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