Summary

Microgavage斑马鱼幼虫

Published: February 20, 2013
doi:

Summary

We present a novel method for microgavage of larval zebrafish utilizing standard embryo microinjection and stereomicroscopy equipment. We demonstrate that microgavage is a safe and efficient technique useful for delivering controlled amounts of diverse materials specifically into the larval zebrafish intestinal lumen.

Abstract

The zebrafish has emerged as a powerful model organism for studying intestinal development1-5, physiology6-11, disease12-16, and host-microbe interactions17-25. Experimental approaches for studying intestinal biology often require the in vivo introduction of selected materials into the lumen of the intestine. In the larval zebrafish model, this is typically accomplished by immersing fish in a solution of the selected material, or by injection through the abdominal wall. Using the immersion method, it is difficult to accurately monitor or control the route or timing of material delivery to the intestine. For this reason, immersion exposure can cause unintended toxicity and other effects on extraintestinal tissues, limiting the potential range of material amounts that can be delivered into the intestine. Also, the amount of material ingested during immersion exposure can vary significantly between individual larvae26. Although these problems are not encountered during direct injection through the abdominal wall, proper injection is difficult and causes tissue damage which could influence experimental results. We introduce a method for microgavage of zebrafish larvae. The goal of this method is to provide a safe, effective, and consistent way to deliver material directly to the lumen of the anterior intestine in larval zebrafish with controlled timing. Microgavage utilizes standard embryo microinjection and stereomicroscopy equipment common to most laboratories that perform zebrafish research. Once fish are properly positioned in methylcellulose, gavage can be performed quickly at a rate of approximately 7-10 fish/ min, and post-gavage survival approaches 100% depending on the gavaged material. We also show that microgavage can permit loading of the intestinal lumen with high concentrations of materials that are lethal to fish when exposed by immersion. To demonstrate the utility of this method, we present a fluorescent dextran microgavage assay that can be used to quantify transit from the intestinal lumen to extraintestinal spaces. This test can be used to verify proper execution of the microgavage procedure, and also provides a novel zebrafish assay to examine intestinal epithelial barrier integrity under different experimental conditions (e.g. genetic manipulation, drug treatment, or exposure to environmental factors). Furthermore, we show how gavage can be used to evaluate intestinal motility by gavaging fluorescent microspheres and monitoring their subsequent transit. Microgavage can be applied to deliver diverse materials such as live microorganisms, secreted microbial factors/toxins, pharmacological agents, and physiological probes. With these capabilities, the larval zebrafish microgavage method has the potential to enhance a broad range of research fields using the zebrafish model system.

Protocol

We use published protocols for standard zebrafish husbandry and maintenance to obtain larvae for microgavage27. We primarily use natural breeding to propagate fish, and they are kept on a 14 hr light/10 hr dark cycle. This protocol has been optimized for microgavage of zebrafish larvae at 6-7 days post-fertilization (dpf). We anticipate that these methods will be generally applicable to younger and older developmental stages with minor modifications. Unless otherwise noted, we used the wild-type TL zebrafish s…

Representative Results

When gavage is performed properly, the delivered material should be entirely contained within the anterior intestine with little to no residual material in the esophagus (Figure 3). A delivery volume should be chosen that can be accommodated within the anterior bulb and does not leak out through cloaca or esophagus. If the volume of material or pressure of delivery is too high, then the physical force of gavage may lead to damage of or leakage through the epithelium. It may also push out or alter other c…

Discussion

In this work, we describe a novel protocol for direct delivery of materials to the larval zebrafish intestine by microgavage. There are several critical steps throughout the procedure that should be kept in mind. First, the zebrafish larvae should be in good health before the gavage experiment to prevent death unrelated to treatment. Another important factor is the quality of the gavage needle. One of the most difficult parts of the protocol is clipping the needle at the appropriate location without creating sharp, jagge…

Divulgations

The authors have nothing to disclose.

Acknowledgements

We thank members of the Rawls laboratory for helpful suggestions on content and Dr. Alan Fanning for valuable discussions on tight junction size permeability and disruption methods. We also thank Dr. Michael Chua and Dr. Neal Kramarcy of the Michael Hooker Microscopy Facility for confocal microscope support. This work was supported by National Institutes of Health grants T32 DK007737-15 (J.L.C. trainee), F32 DK094592 (to J.L.C.), and R01 DK081426 (to J.F.R.).

Materials

Name of the reagent Company Catalogue number Comments
Finquel (Tricaine methanesulfonate) Argent Chemical Laboratories MS-222 Larvae anesthesia
Phenol Red Solution Sigma-Aldrich P0290-100 ml 0.5% in DPBS, cell-culture tested
Mineral oil, light, white (high purity) Amresco J217-500 ml For needle backfill and volume testing
TxRed-Dextran, 10,000 MW, lysine fixable Invitrogen/ Molecular Probes D-1863 1% suspension in 1x PBS/phenol red
FM4-64FX Invitrogen/ Molecular Probes F34653 5 mM stock in water
Methylcellulose MP Biochemicals 0215549590  
Borosilicate glass capillaries Drummond Scientific 3-000-203-G/X OD 1.14 mm, ID 0.53 mm, 3.5 in length
Plastic form for mold making Adaptive Science Tools TU-1  
Nanoject II microinjection unit Drummond Scientific 3-000-204  
Flaming Brown Micropipette Puller, 3.0 mm wide-trough filament Sutter Instrument Co. P-97, FT330B Needle fabrication
Student Dumont #5 forceps Fine Science Tools 91150-20 0.1 x 0.06 mm, needle clipping
Eyepiece Graticle, 5 mm – 100 divisions Leica 10394771 Needle clipping
Microforge Narishige MF-900 Needle clipping and fire-polishing
Tuberculin SlipTip syringe needle Becton Dickinson 309626 1 ml, 25G 5/8-needle
Fluoresbrite YG Microspheres (2.0 μm) Polysciences, Inc. 18338 Intestinal motility analysis
Dissecting Microscope Leica S6E Gavage procedure
Fluorescence Stereomicroscope Leica M205C Dextran barrier assay
Confocal microscope Zeiss LSM510 Imaging of dextran in circulation

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Cocchiaro, J. L., Rawls, J. F. Microgavage of Zebrafish Larvae. J. Vis. Exp. (72), e4434, doi:10.3791/4434 (2013).

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