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 Ram&#243;n 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 Aut&#242;noma de Barcelona for helpful technical assistance.

All experimental procedures involving zebrafish (Danio rerio) were authorized by the Ethics Committee of the Universitat Aut&#242;noma 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&#8211;28 &#176;C.
1. Preparing the Equipment for Oral Intubation
<ol>
	<li>Place approximately 1 cm of a fine silicone tube on a 31 G Luer lock needle to cover the needle tip.</li>
	<li>Cut a 10 &#181;L 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.</li>
	<li>Attach the needle to a 100 &#181;L Luer lock syringe. 
	NOTE: Always rinse with ethanol and then phosphate buffered saline (PBS, see Materials) thoroughly between treatments.</li>
</ol>
2. Solutions Required
<ol>
	<li>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.</li>
	<li>Fill another small tank with 1 L of aquarium water without MS-222 for fish recovery and keep it aerated.</li>
	<li>Make 50 mL of 1x PBS from a 10x sterile stock solution.</li>
	<li>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&#39;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 &#176;C until 30 min before the dissection step.</li>
	<li>For confocal microscopy/sample fixation, prepare 50 mL of fresh 4% paraformaldehyde (PFA) solution in PBS or thaw a stock solution from -20 &#176;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&#160;a fume hood.</li>
</ol>
3. Preparing the Fluorescent Nanoparticle Suspension
<ol>
	<li>Label the protein nanoparticle with Atto-488 NHS ester (see Table of Materials) or an appropriate fluorescent dye according to the manufacturer&#39;s instructions.</li>
	<li>Resuspend the nanoparticles in 0.1 M sodium bicarbonate buffer at the concentration of 2 mg/mL.</li>
	<li>Dissolve the Atto 488 NHS ester in amine-free dimethyl sulfoxide (DMSO) at 2 mg/mL. Keep an aliquot of 10 &#181;L to check labeling efficiency (step 3.7&#8211;3.8).</li>
	<li>Mix the nanoparticles and the Atto 488 NHS ester at a molar ratio of 1:2 (protein:dye) by stirring in the dark.</li>
	<li>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&#8211;3.8).</li>
	<li>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.</li>
	<li>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 &#176;C protected from the light.</li>
	<li>Measure the labeling efficiency using a microvolume spectrophotometer.
	<ol>
		<li>Take 1 &#181;L 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.</li>
		<li>Take 1 &#181;L of the saved supernatant from the labelling reaction in step 3.5. Measure the absorption (abs) at = 501 nm. The percentage of labelling is: 
		(<img alt="Equation 1" src="/files/ftp_upload/58366/58366eq1.jpg" /></li>
	</ol>
	</li>
	<li>Before the experiment, prepare the nanoparticle suspension at the desired concentration using 1x PBS solution.</li>
</ol>
4. Zebrafish Anesthetization and Oral Intubation
<ol>
	<li>Fast the fish (&#62;0.5 g) at least 48 h before the experiment to empty the intestine.</li>
	<li>Move the fish (12 fish) to the experimental tanks (6 L) one night before the experiment to allow acclimatization12.</li>
	<li>Vortex the nanoparticle solution well (e.g.,&#160;2,500 rpm and 30 s) and draw up the desired volume of nanoparticle suspension (e.g.,&#160;20&#8211;50 &#181;L) into the syringe attached to the protected needle.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Slowly inject the nanoparticle suspension to the fish. Make sure the suspension does not flow outward through the gills or mouth.</li>
	<li>Gently remove the needle and place the fish into the recovery tank (see section 2). Recovery usually takes within 1 min.</li>
	<li>Check the fish carefully for any abnormality (e.g., bleeding at the gills is a sign of perforation).</li>
	<li>Once the fish have recovered, return them to the experimental tanks.</li>
</ol>
5. Zebrafish Intestine Dissection
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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 &#60;0.7 g.</li>
	<li>Roll the intestine on the filter paper with tweezers in order to detach the adhesive tissue from the intestine.</li>
	<li>Proceed to prepare the intestine for various downstream analyses (sections 6, 7 and 8).</li>
</ol>
6. Preparing Intestinal Cells for Cytometry
<ol>
	<li>Prepare in advance aliquots of 0.15% collagenase solution (see section 2.4). 
	NOTE: Aliquots should be at room temperature before proceeding.</li>
	<li>Optional:&#160;Continuing from step 5.5, slit open the intestine longitudinally and wash with 1x PBS.</li>
	<li>Using tweezers, place the intestine in the 2 mL centrifuge tube filled with 0.15% collagenase solution.</li>
	<li>Place the tubes on a vertical laboratory rotator for 1 h at room temperature in the dark.</li>
	<li>Place the intestine on a 100 &#181;m 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.</li>
	<li>Centrifuge the 50 mL centrifuge tube at 400 x g for 10 min, at 4 &#176;C.</li>
	<li>Carefully pipette off most of the supernatant while not losing the cells, some of which may be associated to the mucus.</li>
	<li>Resuspend the intestinal cells at the bottom of the centrifuge tube with 500 &#181;L of 1x PBS and keep on ice until cytometry analysis</li>
	<li>Filter samples through a 30 &#181;m cell filter into 5 mL round bottom tubes for cytometry.</li>
	<li>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).</li>
	<li>Immediately analyze the cells on a cytometer, following the instructions of use13.</li>
</ol>
7. Preparing Intestine Cryosections for Confocal Microscopy
<ol>
	<li>Fill the plastic mold (see Table of Materials) to half volume with optimum cutting temperature (O.C.T.) compound.</li>
	<li>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 &#34;Z&#34; shape in O.C.T. compound to easily follow its natural orientation.</li>
	<li>Place the plastic mold on dry ice until it goes opaque (less than a minute).</li>
	<li>Store the plastic mold at -80 &#176;C for long-term use or process immediately using the following procedure. 
	NOTE: The protocol can be paused here.</li>
	<li>Slice the frozen intestine into 10 &#181;m sections or appropriate thickness using a cryostat at -20 &#176;C.</li>
	<li>Collect the intestine section with a fine brush onto a slide.</li>
	<li>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&#160;a fume hood.</li>
	<li>Wash the slide 3 times with 1x PBS, 10 min each.</li>
	<li>Add a drop of mounting medium and place a coverslip over the specimen. 
	NOTE: The protocol can be paused here.</li>
	<li>Observe the sample under a confocal microscope at appropriate magnification.</li>
</ol>
8. Preparing the Intestine for Real Time qPCR (RT-qPCR)
<ol>
	<li>Continuing from step 5.5, put the intestine in a cryogenic vial and rapidly freeze the intestine in liquid nitrogen and store at -80 &#176;C until use. 
	NOTE: The protocol can be paused here.</li>
	<li>For homogenization, add 200 &#181;L of 2% (v/v) chilled 1-Thioglycerol/Homogenization solution (see Table of Materials) or alternative homogenization solution to the intestine sample.</li>
	<li>Work quickly, homogenize the intestine sample on ice with a laboratory homogenizer at high speed (set at 25&#8211;30,000 rpm) until no visible tissue fragments remain. 3 times for&#160;5 s is usually sufficient.</li>
	<li>Isolate RNA using a commercial kit (see Table of Materials) according to the manufacturer&#39;s instructions14 or a suitable alternative method. As needed, store the RNA at -80 &#176;C for long-term use. 
	NOTE: The protocol can be paused here.</li>
	<li>Quantify the RNA concentration using a spectrophotometer15 and evaluate the quality using a RNA analyzer16.</li>
	<li>Prepare 1 &#181;g or an appropriate amount of cDNA using a cDNA synthesis kit according to the manufacturer&#39;s instructions. 
	NOTE: The protocol can be paused here. For qPCR analysis, please follow the MIQE guidelines17.</li>
	<li>Design appropriate primer pairs for the gene/s of interest.</li>
	<li>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 &#181;L of SYBR green supermix, 0.5 &#181;M primers, 2.5 &#181;L of diluted cDNA and 1.5 &#181;L of water in a final volume of 10 &#181;L for each well of qPCR plate.</li>
</ol>

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The authors declare that no competing interests exist.

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...

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 lymphocytes6,7. 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 &#181;L of the protein nanoparticle suspension&#160;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&#39;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.

<table><tbody><tr><td>Silicone tube</td><td>Dow Corning</td><td>508-001</td><td>0.30 mm inner diameter and 0.64 mm outer diameter</td></tr><tr><td>Luer lock needle</td><td>Hamilton</td><td>7750-22</td><td>31 G, Kel-F Hub</td></tr><tr><td>Luer lock syringe</td><td>Hamilton</td><td>81020/01</td><td>100 &amp;mu;L, Kel-F Hub</td></tr><tr><td>Filtered pipette tip</td><td>Nerbe Plus</td><td>07-613-8300</td><td>10 &amp;mu;L</td></tr><tr><td>MS-222</td><td>Sigma Aldrich</td><td>E10521</td><td>powder</td></tr><tr><td>10x PBS</td><td>Sigma Aldrich</td><td>P5493</td><td></td></tr><tr><td>Filter paper&amp;nbsp;</td><td>Filter-Lab</td><td>RM14034252</td><td></td></tr><tr><td>Collagenase</td><td>Gibco</td><td>17104019</td><td></td></tr><tr><td>DMEM&amp;nbsp;</td><td>Gibco</td><td>31966</td><td>Dulbecco&#039;s modified eagle medium</td></tr><tr><td>Penicillin and streptomycin</td><td>Gibco</td><td>15240</td><td></td></tr><tr><td>Cell strainer</td><td>Falcon</td><td>352360</td><td></td></tr><tr><td>CellTrics filters&amp;nbsp;</td><td>Sysmex Partec</td><td>04-004-2326 (Wolflabs)</td><td>30 &amp;micro;m mesh size filters with 2 mL reservoir</td></tr><tr><td>Tissue-Tek O.C.T. compound</td><td>SAKURA</td><td>4583</td><td></td></tr><tr><td>Plastic molds for cryosections</td><td>SAKURA</td><td>4557</td><td>Disposable Vinyl molds. 25 mm x 20 mm x 5 mm</td></tr><tr><td>Slide</td><td>Thermo Scientific</td><td>10149870</td><td>SuperFrost Plus slide</td></tr><tr><td>Cover glasses</td><td>Labbox&amp;nbsp;</td><td>COVN-024-200</td><td>24&amp;acute;24 mm</td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Sigma-Aldrich</td><td>158127</td><td></td></tr><tr><td>Atto-488 NHS ester</td><td>Sigma-Aldrich</td><td>41698</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Sigma-Aldrich</td><td>S5761</td><td></td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>D8418</td><td></td></tr><tr><td>Maxwell RSC simplyRNA Tissue Kit</td><td>Promega</td><td>AS1340</td><td></td></tr><tr><td>1-Thioglycerol/Homogenization solution</td><td>Promega</td><td>Inside of Maxwell RSC simplyRNA Tissue Kit</td><td>adding 20 &amp;mu;l 1-Thioglycerol to 1 mL homogenization solution (2%)</td></tr><tr><td>vertical laboratory rotator&amp;nbsp;</td><td>Suministros Grupo Esper</td><td>10000-01062</td><td></td></tr><tr><td>Cryostat</td><td>Leica&amp;nbsp;</td><td>CM3050S</td><td></td></tr><tr><td>Homogenizer</td><td>KINEMATICA</td><td>Polytron PT1600E</td><td></td></tr><tr><td>Flow cytometer&amp;nbsp;</td><td>Becton Dickinson</td><td>FACS Canto</td><td></td></tr><tr><td>5 mL round bottom tube</td><td>Falcon</td><td>352058</td><td></td></tr><tr><td>Confocal microscope</td><td>Leica</td><td>SP5</td><td></td></tr><tr><td>Fume Hood</td><td>Kottermann</td><td>2-447 BST</td><td></td></tr><tr><td>Nanodrop 1000</td><td>Thermo Fisher Scientific</td><td>ND-1000</td><td>Spectrophotometer</td></tr><tr><td>Agilent 2100 Bioanalyzer System</td><td>Agilent</td><td>G2939A</td><td>RNA bioanalyzer</td></tr><tr><td>Maxwell Instrument</td><td>Promega</td><td>AS4500&amp;nbsp;</td><td></td></tr><tr><td>iScript cDNA synthesis kit&amp;nbsp;</td><td>Bio-rad</td><td>1708891</td><td></td></tr><tr><td>CFX384 Real-Time PCR&amp;nbsp;Detection System</td><td>Bio-Rad</td><td>1855485</td><td></td></tr><tr><td>iTaq universal SYBR Green Supermix kit</td><td>Bio-rad</td><td>172-5120</td><td></td></tr><tr><td>Water&amp;nbsp;</td><td>Sigma-Aldrich</td><td>W4502</td><td></td></tr><tr><td>Cryogenic vial&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>375418</td><td>CryoTube vial</td></tr><tr><td>Mounting medium</td><td>Sigma-Aldrich</td><td>F6057</td><td>Fluoroshield with DAPI</td></tr></tbody></table>

oral intubation of adult zebrafish: a model for evaluating intestinal uptake of bioactive compounds

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 &#181;L 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.

Zebrafish (average weight: 1.03 &#177; 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 &#181;L or 50 &#181;L of nanoparticle suspensions and the mortality ...

Watch this Scientific Journal Video about Oral Intubation of Adult Zebrafish: A Model for Evaluating Intestinal Uptake of Bioactive Compounds at JoVE.com

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

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&#160;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 ...

oral-intubation-adult-zebrafish-model-for-evaluating-intestinal

Research

JoVE Journal

Immunology and Infection

7.8K Views.  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.

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

Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their intercommunication, are paramount for the protection against infections. The interactions of epithelial and innate immune cells with a pathogen are best investigated in vivo, where complex behavior unfolds over time and space. However, existing models do not allow for easy spatio-temporal imaging of the battle with pathogens at the mucosal level.
The model developed here creates a mucosal infection by direct injection of the fungal pathogen, Candida albicans, into the swimbladder of juvenile zebrafish. The resulting infection enables high-resolution imaging of epithelial and innate immune cell behavior throughout the development of mucosal disease. The versatility of this method allows for interrogation of the host to probe the detailed sequence of immune events leading to phagocyte recruitment and to examine the roles of particular cell types and molecular pathways in protection. In addition, the behavior of the pathogen as a function of immune attack can be imaged simultaneously by using fluorescent protein-expressing C. albicans. Increased spatial resolution of the host-pathogen interaction is also possible using the described rapid swimbladder dissection technique.
The mucosal infection model described here is straightforward and highly reproducible, making it a valuable tool for the study of mucosal candidiasis. This system may also be broadly translatable to other mucosal pathogens such as mycobacterial, bacterial or viral microbes that normally infect through epithelial surfaces.

In vivo spatio-temporal interactions of pathogen and immune defenses at the mucosal level are not easily imaged in existing vertebrate hosts. The method presented here describes a versatile platform to study mucosal candidiasis in live vertebrates using the swimbladder of the juvenile zebrafish as an infection site.

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their ...

Electrophysiological Recording in the Brain of Intact Adult Zebrafish

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram recordings. This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments, allowing recording of neural activity. Immobilization of the adult requires a mechanism to deliver dissolved oxygen to the gills in lieu of buccal and opercular movement. With our technique, animals are immobilized and perfused with habitat water to fulfill this requirement. A craniotomy is performed under tricaine methanesulfonate (MS-222; tricaine) anesthesia to provide access to the brain. The primary electrode is then positioned within the craniotomy window to record extracellular brain activity. Through the use of a multitube perfusion system, a variety of pharmacological compounds can be administered to the adult fish and any alterations in the neural activity can be observed. The methodology not only allows for observations to be made regarding changes in neurological activity, but it also allows for comparisons to be made between larval and adult zebrafish. This gives researchers the ability to identify the alterations in neurological activity due to the introduction of various compounds at different life stages.

This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments to allow recordings and manipulation of neural activity in an intact animal.

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram ...

Neuroscience

Intraperitoneal Injection into Adult Zebrafish

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, this method makes it difficult to know how much reagent is absorbed or taken up per fish. Some experimental questions, particularly those related to metabolic studies, may be better addressed by delivering a defined quantity to each fish, based on weight. Here we present a method for intraperitoneal (IP) injection into adult zebrafish. Injection is into the abdominal cavity, posterior to the pelvic girdle. This procedure is adapted from veterinary methods used for larger fish. It is safe, as we have observed zero mortality. Additionally, we have seen bleeding at the injection site in only 5 out of 127 injections, and in each of those cases the bleeding was brief, lasting several seconds, and the quantity of blood lost was small. Success with this procedure requires gentle handling of the fish through several steps including fasting, weighing, anesthetizing, injection, and recovery. Precautions are required to minimize stress throughout the procedure. Our precautions include using a small injection volume and a 35G needle. We use Cortland salt solution as the vehicle, which is osmotically balanced for freshwater fish. Aeration of the gills is maintained during the injection procedure by first bringing the fish into a surgical plane of anesthesia, which allows slow operculum movements, and second, by holding the fish in a trough within a water-saturated sponge during the injection itself. We demonstrate the utility of IP injection by injecting glucose and monitoring the rise in blood glucose level and its subsequent return to normal. As stress is known to increase blood glucose in teleost fish, we compare blood glucose levels in vehicle-injected and non-injected adults and show that the procedure does not cause a significant rise in blood glucose.

We demonstrate intraperitoneal injection into adult zebrafish. We use a 10 &mu;l NanoFil microsyringe controlled by a Micro4 controller and UltraMicroPump III. This demonstration includes the use of cold water as an anesthetic.

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, ...

Biology

Optimized Intravenous Injection in Adult Zebrafish

Intravenous (IV) injection is widely recognized as the most effective and commonly utilized method for achieving systemic delivery of substances in mammalian research models. However, its application in adult zebrafish for drug delivery, stem cell transplantation, and regenerative and cancer studies has been limited due to the challenges posed by their small body size and intricate blood vessels. To overcome these limitations, alternative injection techniques such as intracardiac and retro-orbital (RO) injection have been explored in the past for stem cell transplantation in adult zebrafish. However, these techniques have their drawbacks, including the need for meticulous injection techniques or increased risk of mortality.
In this study, we have developed a refined and optimized IV injection procedure specifically tailored to adult zebrafish, addressing the challenges associated with their unique anatomy. To demonstrate the effectiveness of this technique, we performed successful IV injections of whole kidney marrow cells from Tg(mpo: EGFP) fish and FITC-dextran dye into adult Casper fish. The subsequent visualization of injected cells and dyes using a fluorescence microscope confirmed their successful delivery and engraftment within the zebrafish. Furthermore, we demonstrated that compared with the intracardiac and RO injections, the IV injection resulted in improved survival rates and engraftment efficiency in treated zebrafish. This approach enables precise delivery and localization of substances and holds great potential for large-scale drug and chemical screening using adult zebrafish. Additionally, the ability to visually track the injected cells and dyes provides invaluable insights into their engraftment, migration, and interactions with host tissues, enabling a more comprehensive evaluation of therapeutic effects and biological processes in zebrafish models.

Here, we present an optimized and effective protocol for the intravenous injection of cells or pharmacological agents into adult zebrafish, resulting in enhanced cell engraftment and increased survival rates of the treated zebrafish.

Intravenous (IV) injection is widely recognized as the most effective and commonly utilized method for achieving systemic delivery of substances in ...

Cancer Research

Microgavage of Zebrafish Larvae

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.

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.

The zebrafish has emerged as a powerful model organism for studying intestinal development1-5, physiology6-11, disease12-16, and host-microbe ...

A Doxorubicin-induced Cardiomyopathy Model in Adult Zebrafish

The genetically accessible adult zebrafish (Danio rerio) has been increasingly used as a vertebrate model for understanding human diseases such as cardiomyopathy. Because of its convenience and amenability to high throughput genetic manipulations, the generation of acquired cardiomyopathy models, such as the doxorubicin-induced cardiomyopathy (DIC) model in adult zebrafish, is opening the doors to new research avenues, including discovering cardiomyopathy modifiers via forward genetic screening. Different from the embryonic zebrafish DIC model, both initial acute and later chronic phases of cardiomyopathy can be determined in the adult zebrafish DIC model, enabling the study of stage-dependent signaling mechanisms and therapeutic strategies. However, variable results can be obtained with the current model, even in the hands of experienced investigators. To facilitate future implementation of the DIC model, we present a detailed protocol on how to generate this DIC model in adult zebrafish and describe two alternative ways of intraperitoneal (IP) injection. We further discuss options on how to reduce variations to obtain reliable results and provide suggestions on how to appropriately interpret the results.

A method to generate a doxorubicin-induced cardiomyopathy model in adult zebrafish (Danio rerio) is described here. Two alternative ways of intraperitoneal injection are presented and conditions to reduce variations among different experimental groups are discussed.

The genetically accessible adult zebrafish (Danio rerio) has been increasingly used as a vertebrate model for understanding human diseases such as ...

Medicine

Gavaging Adult Zebrafish

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 &mu;l of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.

The increasing use of zebrafish as an animal model requires the development of effective methods for the delivery of known quantities of compounds and solutions. The gavage procedure described below allows for the oral delivery of precise volumes of solution reliably, safely and efficiently to adult zebrafish.

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or ...

High-throughput Measurement of Gut Transit Time Using Larval Zebrafish

Zebrafish are used as alternative model organisms for drug safety testing. The gastrointestinal (GI) tract of zebrafish has genetic, neuronal, and pharmacological similarities to that of the mammals. GI intolerance during clinical testing of drug candidates is common and may pose a serious threat to human health. Testing for GI toxicity in preclinical mammalian models can be expensive in terms of time, test compound, and labor. The high-throughput method presented here may be used to predict GI safety issues. Compared to mammalian models, this method allows for more expedient assessment of test compound effects on GI transit while using low quantities of compound. In this method, larval zebrafish (7 days post fertilization) are fed food containing a fluorescent label. After feeding, each larval fish is placed into a well of a 96-conical-bottom-well plate and dosed with test compound (dissolved in water) or the vehicle. As gut transit occurs, fecal matter accumulates on the bottom of the wells, and the rate at which this happens is monitored by measuring fluorescence from the bottom of the well repeatedly over time using a plate spectrophotometer. The fluorescence from larvae in a given treatment group are averaged and these values are graphed along with standard error, for each measurement time, yielding a curve representing average transit of food over time. Effects on gut transit time are identified by comparing the area under the curve for each treatment group to that of the vehicle-treated group. This method detected changes in zebrafish GI transit time induced by drugs with known clinical GI effects; it can be employed to interrogate dozens of treatments for GI effects per day. As such, safer compounds can be quickly prioritized for mammalian testing, which expedites discovery and proffers 3Rs advancement.

The goal of this protocol is to measure the transit time of fluorescently labeled food through the gut of larval zebrafish in a high throughput fashion.

Zebrafish are used as alternative model organisms for drug safety testing. The gastrointestinal (GI) tract of zebrafish has genetic, neuronal, and ...

Optimized Intraperitoneal Injection for Compound Delivery in Adult Zebrafish

An Intraperitoneal Injection Technique in Adult Zebrafish that Minimizes Body Damage and Associated Mortality

The adult zebrafish (Danio rerio), which is genetically accessible, is being employed as a valuable vertebrate model to study human disorders such as cardiomyopathy. Intraperitoneal (IP) injection is an important method that delivers compounds to the body for either testing therapeutic effects or generating disease models such as doxorubicin-induced cardiomyopathy (DIC). Currently, there are two methods of IP injection. Both&#160;methods have limitations when handling toxic compounds such as doxorubicin, which result in side effects manifesting as severe damage to the body shape and fish death. While these shortcomings could be overcome by extensive investigator training, a new IP injection method that has minimal side effects is desirable. Here, a unique IP injection method that is able to handle toxic compounds is reported. Consistently reduced cardiac function can result without incurring significant fish death. The technique can be easily mastered by researchers who have minimal experience with adult zebrafish.

Author Spotlight: Novel Intraperitoneal Injection Method for Zebrafish Cardiotoxicity Models

A new intraperitoneal (IP) injection method in adult zebrafish is described. When handling toxic compounds such as doxorubicin, this procedure is more effective than the two previously reported IP methods. The technique is designed to be easily adopted by researchers with limited experience in the zebrafish model.

The adult zebrafish (Danio rerio), which is genetically accessible, is being employed as a valuable vertebrate model to study human disorders such as ...

Oral Intubation of Adult Zebrafish to Administer Experimental Compounds

Source: Ji, J., Thwaite, R. et al. <a href="https://app.jove.com/v/58366">Oral Intubation of Adult Zebrafish: A Model for Evaluating Intestinal Uptake of Bioactive Compounds.</a> J. Vis. Exp. (2018).
This video demonstrates a technique for orally intubating adult zebrafish to administer experimental compounds into the esophagus and monitor their intestinal uptake. This method offers a valuable model for studying the dynamics of oral prophylactics or other compounds within the intestine.