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 tract of adult zebrafish². In addition, Chen et al. used zebrafish oral intubation to show that drugs encapsulated by nanoparticles, when administered via the gastro-intestinal tract, were transported across the blood brain barrier³. These authors performed intubation based on the gauvage method described by Collymore et al.⁴ with some modifications. However, they did not provide a highly detailed protocol describing the oral intubation procedure. Here, we present a method for oral intubation of adult zebrafish building on Collymore et al.⁴ We further include the preparation of the intestine for relevant downstream analysis by cytometry, confocal microscopy and qPCR.

The intestine and particularly its mucosa is the first-line of defense against infection and the primary site of nutrient uptake⁵. When the epithelial cells and antigen-presenting cells within mucosal barriers perceive danger signals, an immediate innate immune response is triggered. Next, the highly specific adaptive immune response is established by T and B lymphocytes^6,7. Development of oral vaccines is a current focus area in vaccinology. Such vaccines would be an effective tool to protect the organism at exposed sites due to the specific response of immune cells in the mucosa-associated lymphoid tissues (MALT)^8,9. In aquaculture, mucosal vaccines have obvious advantages compared to injectable vaccines. They are practical for mass vaccination, less labor-intensive, are less stressful to the fish, and can be administered to young fish. Nevertheless, mucosal vaccine candidates must reach the second gut segment without being denatured in the oral environment. They also must cross mucosal barriers in order to gain access to antigen presenting cells (APCs) to induce local and/or systemic responses¹⁰. Hence, testing of the mucosal uptake achieved by candidate oral antigens and their delivery systems, as well as the immune response evoked, is essential in the development of oral vaccines.

In a biomedical context, developing a model to test biological effects of compounds after oral intubation is of growing interest. Many of the anatomical and physiological features of the intestine are conserved between bilaterian lineages, with mammals and bony fishes¹¹. This oral intubation model connected to downstream analysis can be a tool to provide insights into human biology, as well as a testing ground for biologics or other compounds in vivo.

The oral intubation protocol can be performed by one operator, e.g., successfully administrating up to 50 µL of the protein nanoparticle suspension to fish weighing 1 g, with a high survival rate. The procedure is simple to set up and quick; 30 fish can be intubated in 1 h. The protocol for intestine preparation is key to providing quality cell and tissue samples for subsequent analysis. Examples of downstream results are given which show the protocol's usefulness in obtaining data related to intestinal uptake and in isolating quality RNA for qPCR. The protocol would be of great use to those needing a suitable model to test the dynamics of oral prophylactics or other compounds in the intestine.

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. Make sure the pipette extends beyond the tip of the needle to avoid injuring the animal.
3. Attach the needle to a 100 µL Luer lock syringe.
   NOTE: Always rinse with ethanol and then phosphate buffered saline (PBS, see Materials) thoroughly between treatments.

2. Solutions Required

1. Prepare 150 mg/L (for anesthesia) or 300 mg/L (for euthanasia) of ethyl 3-aminobenzoate methanesulfonate (MS-222) solution with water from the aquarium where the zebrafish are maintained. Fill a small tank with 1 L of anesthetic solution and keep it aerated.
2. Fill another small tank with 1 L of aquarium water without MS-222 for fish recovery and keep it aerated.
3. Make 50 mL of 1x PBS from a 10x sterile stock solution.
4. For cytometry analysis/intestinal cell isolation, prepare enough fresh 0.15% collagenase Type IV solution for 1 mL per fish from a stock solution or from powder in Dulbecco's modified eagle medium (DMEM) with 1% v/v penicillin and streptomycin (see materials). Make aliquots (1 per fish) of 1 mL in 2 mL centrifuge tubes. Keep at 4 °C until 30 min before the dissection step.
5. For confocal microscopy/sample fixation, prepare 50 mL of fresh 4% paraformaldehyde (PFA) solution in PBS or thaw a stock solution from -20 °C freezer in a fume hood.
   CAUTION: PFA is toxic. Please read the material safety data sheet before working with it. Gloves and safety glasses should be worn, and always leave solutions inside a fume hood.

3. Preparing the Fluorescent Nanoparticle Suspension

1. Label the protein nanoparticle with Atto-488 NHS ester (see Table of Materials) or an appropriate fluorescent dye according to the manufacturer's instructions.
2. Resuspend the nanoparticles in 0.1 M sodium bicarbonate buffer at the concentration of 2 mg/mL.
3. Dissolve the Atto 488 NHS ester in amine-free dimethyl sulfoxide (DMSO) at 2 mg/mL. Keep an aliquot of 10 µL to check labeling efficiency (step 3.7–3.8).
4. Mix the nanoparticles and the Atto 488 NHS ester at a molar ratio of 1:2 (protein:dye) by stirring in the dark.
5. Spin down the labeled nanoparticles by centrifugation at 8,000 x g for 10 min at room temperature, remove the supernatant and keep it to check labeling efficiency (step 3.7–3.8).
6. Wash the labeled nanoparticles by resuspending in 1 mL of 0.1 M sodium bicarbonate buffer by vortexing and pipetting up and down. Then discard the supernatant by centrifugation at 8,000 x g for 10 min at room temperature. Repeat step 3.6 for 5 times.
7. Resuspend the pellet in 5 mL of 0.1 M sodium bicarbonate buffer in a 15 mL centrifuge tube and make aliquots of the fluorescent nanoparticle in 1.5 mL centrifuge tubes (30 aliquots). Spin down at 8,000 x g for 10 min at room temperature, discard the supernatant and store at -80 °C protected from the light.
8. Measure the labeling efficiency using a microvolume spectrophotometer.
   1. Take 1 µL of the original Atto 488 solution kept from step 3.3 and further dilute it in DMSO (e.g., 1:20 according to the volume ratio). This is the volume used to get the molar ratio of the protein nanoparticle and Atto 488 mix at step 3.4.
   2. Take 1 µL of the saved supernatant from the labelling reaction in step 3.5. Measure the absorption (abs) at = 501 nm. The percentage of labelling is:
      (
9. Before the experiment, prepare the nanoparticle suspension at the desired concentration using 1x PBS solution.

4. Zebrafish Anesthetization and Oral Intubation

1. Fast the fish (>0.5 g) at least 48 h before the experiment to empty the intestine.
2. Move the fish (12 fish) to the experimental tanks (6 L) one night before the experiment to allow acclimatization¹².
3. Vortex the nanoparticle solution well (e.g., 2,500 rpm and 30 s) and draw up the desired volume of nanoparticle suspension (e.g., 20–50 µL) into the syringe attached to the protected needle.
4. Place the fish in the aerated 150 mg/L MS-222 solution (see section 2) until they sink to the bottom of the tank and do not respond to a tail fin pinch; this process takes less than 5 min.
5. Quickly transfer the anesthetized fish with a net to a wet plastic tray, orientate the animal horizontally to face the needle and immediately start the oral intubation.
6. Carefully support the fish with one hand and open the mouth with the other hand using the protected needle. Gently insert the needle down the esophagus to about 1 cm from the mouth opening.
   NOTE: The operator may feel a slight resistance when the end of the pipette tip has passed the gill. Take care not to angle the needle entry too much which may perforate the gill.
7. Slowly inject the nanoparticle suspension to the fish. Make sure the suspension does not flow outward through the gills or mouth.
8. Gently remove the needle and place the fish into the recovery tank (see section 2). Recovery usually takes within 1 min.
9. Check the fish carefully for any abnormality (e.g., bleeding at the gills is a sign of perforation).
10. Once the fish have recovered, return them to the experimental tanks.

5. Zebrafish Intestine Dissection

1. After a specified period of time post intubation (e.g., 5 h and/or 24 h), place the fish using a net into 300 mg/L MS-222 solution for euthanasia (see section 2). Make sure the operculum stops moving and there is no tail pinch reflex. Five minutes is normally sufficient.
2. Pick up the euthanized animal with a net and place it on a filter paper.
   NOTE: The filter paper is very useful for removing the adhesive tissue along the intestine.
3. Using sharp dissection scissors, make a semi-circular incision from the anus to the operculum and open incision using fine tweezers. Cut both ends of the intestine, take out all the internal organs, and place them on the filter paper.
   CAUTION: Work quickly to reduce cell metabolism and death.
   NOTE: Alternatively, remove the adhesive tissue in PBS and on ice.
4. Separate the intestine from internal organs making sure to keep its orientation (anterior to posterior intestinal segment) and stretch it out. Usually, the anterior segment of intestine is wider than posterior segment. Take care to obtain all of the intestine when dissecting.
   NOTE: The posterior end is quite fine and fragile in small fish and may break off, particularly in animals <0.7 g.
5. Roll the intestine on the filter paper with tweezers in order to detach the adhesive tissue from the intestine.
6. Proceed to prepare the intestine for various downstream analyses (sections 6, 7 and 8).

6. Preparing Intestinal Cells for Cytometry

1. Prepare in advance aliquots of 0.15% collagenase solution (see section 2.4).
   NOTE: Aliquots should be at room temperature before proceeding.
2. Optional: Continuing from step 5.5, slit open the intestine longitudinally and wash with 1x PBS.
3. Using tweezers, place the intestine in the 2 mL centrifuge tube filled with 0.15% collagenase solution.
4. Place the tubes on a vertical laboratory rotator for 1 h at room temperature in the dark.
5. Place the intestine on a 100 µm cell strainer supported over a 50 mL centrifuge tube. Break up the intestine with a 5 mL syringe plunger, washing 3 times with 1x PBS, collecting the flow through sample in the 50 mL centrifuge tube.
6. Centrifuge the 50 mL centrifuge tube at 400 x g for 10 min, at 4 °C.
7. Carefully pipette off most of the supernatant while not losing the cells, some of which may be associated to the mucus.
8. Resuspend the intestinal cells at the bottom of the centrifuge tube with 500 µL of 1x PBS and keep on ice until cytometry analysis
9. Filter samples through a 30 µm cell filter into 5 mL round bottom tubes for cytometry.
10. Set the parameters (e.g., the number of cells for analysis, the region of interest, voltage and compensation, selection of detectors) on a cytometer equipment (see materials).
11. Immediately analyze the cells on a cytometer, following the instructions of use¹³.

7. Preparing Intestine Cryosections for Confocal Microscopy

1. Fill the plastic mold (see Table of Materials) to half volume with optimum cutting temperature (O.C.T.) compound.
2. Continuing from step 5.5 and immediately after the dissection, carefully place the intestine in the plastic mold. Make sure the intestine is totally embedded in the O.C.T. compound. If necessary, add more O.C.T. compound to the plastic mold.
   NOTE: It is recommended to place the intestine with a "Z" shape in O.C.T. compound to easily follow its natural orientation.
3. Place the plastic mold on dry ice until it goes opaque (less than a minute).
4. Store the plastic mold at -80 °C for long-term use or process immediately using the following procedure.
   NOTE: The protocol can be paused here.
5. Slice the frozen intestine into 10 µm sections or appropriate thickness using a cryostat at -20 °C.
6. Collect the intestine section with a fine brush onto a slide.
7. Immerse the slide in 4% PFA for 15 min at room temperature to fix the sample.
   CAUTION: PFA is toxic. Please read the material safety data sheet before working with it. Gloves and safety glasses should be worn, and always leave solutions inside a fume hood.
8. Wash the slide 3 times with 1x PBS, 10 min each.
9. Add a drop of mounting medium and place a coverslip over the specimen.
   NOTE: The protocol can be paused here.
10. Observe the sample under a confocal microscope at appropriate magnification.

8. Preparing the Intestine for Real Time qPCR (RT-qPCR)

1. Continuing from step 5.5, put the intestine in a cryogenic vial and rapidly freeze the intestine in liquid nitrogen and store at -80 °C until use.
   NOTE: The protocol can be paused here.
2. For homogenization, add 200 µL of 2% (v/v) chilled 1-Thioglycerol/Homogenization solution (see Table of Materials) or alternative homogenization solution to the intestine sample.
3. Work quickly, homogenize the intestine sample on ice with a laboratory homogenizer at high speed (set at 25–30,000 rpm) until no visible tissue fragments remain. 3 times for 5 s is usually sufficient.
4. Isolate RNA using a commercial kit (see Table of Materials) according to the manufacturer's instructions¹⁴ or a suitable alternative method. As needed, store the RNA at -80 °C for long-term use.
   NOTE: The protocol can be paused here.
5. Quantify the RNA concentration using a spectrophotometer¹⁵ and evaluate the quality using a RNA analyzer¹⁶.
6. Prepare 1 µg or an appropriate amount of cDNA using a cDNA synthesis kit according to the manufacturer's instructions.
   NOTE: The protocol can be paused here. For qPCR analysis, please follow the MIQE guidelines¹⁷.
7. Design appropriate primer pairs for the gene/s of interest.
8. Select a suitable reference gene and analyze the expression of each gene by a RT-qPCR detection system using a commercial kit (see materials).
   NOTE: For example, add 5 µL of SYBR green supermix, 0.5 µM primers, 2.5 µL of diluted cDNA and 1.5 µL of water in a final volume of 10 µL for each well of qPCR plate.

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