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...

<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 border="0" cellpadding="0" cellspacing="0" style="width:1271px;" width="1270">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Silicon tube</td>
			<td style="width:227px;">Dow Corning</td>
			<td style="width:295px;">508-001</td>
			<td style="width:460px;">0.30 mm inner diameter and 0.64 mm outer diameter</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Luer lock needle</td>
			<td style="width:227px;">Hamilton</td>
			<td style="width:295px;">7750-22</td>
			<td style="width:460px;">31 G, Kel-F Hub</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Luer lock syringe</td>
			<td style="width:227px;">Hamilton</td>
			<td style="width:295px;">81020/01</td>
			<td style="width:460px;">100 &#956;L, Kel-F Hub</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Filtered pipette tip</td>
			<td style="width:227px;">Nerbe Plus</td>
			<td style="width:295px;">07-613-8300</td>
			<td style="width:460px;">10 &#956;L</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">MS-222</td>
			<td style="width:227px;">Sigma Aldrich</td>
			<td style="width:295px;">E10521</td>
			<td style="width:460px;">powder</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">10x PBS</td>
			<td style="width:227px;">Sigma Aldrich</td>
			<td style="width:295px;">P5493</td>
			<td style="width:460px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Filter paper&#160;</td>
			<td style="width:227px;">Filter-Lab</td>
			<td style="width:295px;">RM14034252</td>
			<td style="width:460px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Collagenase</td>
			<td style="width:227px;">Gibco</td>
			<td style="width:295px;">17104019</td>
			<td style="width:460px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">DMEM&#160;</td>
			<td style="width:227px;">Gibco</td>
			<td style="width:295px;">31966</td>
			<td style="width:460px;">Dulbecco&#39;s modified eagle medium</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Penicillin and streptomycin</td>
			<td style="width:227px;">Gibco</td>
			<td style="width:295px;">15240</td>
			<td style="width:460px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Cell strainer</td>
			<td style="width:227px;">Falcon</td>
			<td style="width:295px;">352360</td>
			<td style="width:460px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">CellTrics filters&#160;</td>
			<td style="width:227px;">Sysmex Partec</td>
			<td style="width:295px;">04-004-2326 (Wolflabs)</td>
			<td style="width:460px;">30 &#181;m mesh size filters with 2 mL reservoir</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Tissue-Tek O.C.T. compound</td>
			<td style="width:227px;">SAKURA</td>
			<td style="width:295px;">4583</td>
			<td style="width:460px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">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 height="22">
			<td height="22" style="height:22px;width:289px;">Slide</td>
			<td style="width:227px;">Thermo Scientific</td>
			<td style="width:295px;">10149870</td>
			<td style="width:460px;">SuperFrost Plus slide</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Cover glasses</td>
			<td style="width:227px;">Labbox&#160;</td>
			<td style="width:295px;">COVN-024-200</td>
			<td style="width:460px;">24&#180;24 mm</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Paraformaldehyde (PFA)</td>
			<td style="width:227px;">Sigma-Aldrich</td>
			<td style="width:295px;">158127</td>
			<td style="width:460px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Atto-488 NHS ester</td>
			<td style="width:227px;">Sigma-Aldrich</td>
			<td style="width:295px;">41698</td>
			<td style="width:460px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Sodium bicarbonate</td>
			<td style="width:227px;">Sigma-Aldrich</td>
			<td style="width:295px;">S5761</td>
			<td style="width:460px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">DMSO</td>
			<td style="width:227px;">Sigma-Aldrich</td>
			<td style="width:295px;">D8418</td>
			<td style="width:460px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Maxwell RSC simplyRNA Tissue Kit</td>
			<td style="width:227px;">Promega</td>
			<td style="width:295px;">AS1340</td>
			<td style="width:460px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:289px;">1-Thioglycerol/Homogenization solution</td>
			<td style="width:227px;">Promega</td>
			<td style="width:295px;">Inside of Maxwell RSC simplyRNA Tissue Kit</td>
			<td style="width:460px;">adding 20 &#956;l 1-Thioglycerol to 1 ml homogenization solution (2%)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">vertical laboratory rotator&#160;</td>
			<td style="width:227px;">Suministros Grupo Esper</td>
			<td style="width:295px;">10000-01062</td>
			<td style="width:460px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Cryostat</td>
			<td style="width:227px;">Leica&#160;</td>
			<td style="width:295px;">CM3050S</td>
			<td style="width:460px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Homogenizer</td>
			<td style="width:227px;">KINEMATICA</td>
			<td style="width:295px;">Polytron PT1600E</td>
			<td style="width:460px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Flow cytometer&#160;</td>
			<td style="width:227px;">Becton Dickinson</td>
			<td style="width:295px;">FACS Canto</td>
			<td style="width:460px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">5 mL round bottom tube</td>
			<td style="width:227px;">Falcon</td>
			<td style="width:295px;">352058</td>
			<td style="width:460px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Confocal microscope</td>
			<td style="width:227px;">Leica</td>
			<td style="width:295px;">SP5</td>
			<td style="width:460px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Fume Hood</td>
			<td style="width:227px;">Kottermann</td>
			<td style="width:295px;">2-447 BST</td>
			<td style="width:460px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Nanodrop 1000</td>
			<td style="width:227px;">Thermo Fisher Scientific</td>
			<td style="width:295px;">ND-1000</td>
			<td style="width:460px;">Spectrophotometer</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Agilent 2100 Bioanalyzer System</td>
			<td style="width:227px;">Agilent</td>
			<td style="width:295px;">G2939A</td>
			<td>RNA bioanalyzer</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Maxwell Instrument</td>
			<td style="width:227px;">Promega</td>
			<td style="width:295px;">AS4500&#160;</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">iScript cDNA synthesis kit&#160;</td>
			<td style="width:227px;">Bio-rad</td>
			<td style="width:295px;">1708891</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:22px;width:289px;">CFX384 Real-Time PCR&#160;Detection System</td>
			<td style="width:227px;">Bio-Rad</td>
			<td style="width:295px;">1855485</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:289px;">iTaq universal SYBR Green Supermix kit</td>
			<td style="width:227px;">Bio-rad</td>
			<td style="width:295px;">172-5120</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Water&#160;</td>
			<td style="width:227px;">Sigma-Aldrich</td>
			<td style="width:295px;">W4502</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Cryogenic vial&#160;</td>
			<td style="width:227px;">Thermo Fisher Scientific</td>
			<td style="width:295px;">375418</td>
			<td>CryoTube vial</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Mounting medium</td>
			<td style="width:227px;">Sigma-Aldrich</td>
			<td style="width:295px;">F6057</td>
			<td>Fluoroshield with DAPI</td>
		</tr>
	</tbody>
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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 ...

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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 ...

This method can help answer key questions in the mucosa vaccinology field, such as how intestinal mucosa uptake biological compounds and how such biologicals modulate the immune system at local site after intubation. The main advantage of this technique is that it's simple to set up, quick, and shows low mortality rates. This method can provide insight into mucosal vaccinology.It can also be applied to other compounds, such as chemicals, toxics, or pathogens. Visual demonstration of this method is critical as in the oral intubation and intestinal dissection steps, you need to be careful because in intubation you could injure the zebrafish and cause death. In dissection, the end of the intestine is very fine and fragile.Place approximately one centimeter of fine silicone tubing on a 31-gauge Luer-lock needle. Cut a sterile 10-microliter filter pipette tip, take the finer end, and place it over the silicone tube as a sheath. To begin, attach the needle apparatus to a 100-microliter Luer-lock syringe.First, move the fish to the experimental tanks one night prior to the experiment. Vortex the prepared nanoparticle solution well, and draw up the desired volume of the suspension. After safely anesthetizing the fish, use a net to quickly transfer the fish to a wet plastic tray.Orient the animal horizontally to face the needle. Carefully support the fish with one hand, and open the mouth with the protected needle. Then, gently insert the needle down the esophagus to about one centimeter from the mouth opening.It's difficult to see how far the needle should go, and this is a matter of practice. You will feel the needle as it finds a stop, and then by manipulating the needle, you'll find the correct angle to insert the device a little more. Slowly inject the nanoparticle suspension into the fish, ensuring the suspension does not flow outward through the gills or the mouth.After this, gently remove the needle, and place the fish into a recovery tank. While the fish recovers, monitor the fish for any abnormalities. Once the fish has recovered, return it to one of the experimental tanks.After euthanizing the fish according to the text protocol, place the fish on a piece of filter paper. Using sharp dissection scissors, make a semi-circular incision from the anus to the operculum, and open the incision with fine tweezers. Then, cut both ends of the intestine, take out all of the internal organs, and place them on the filter paper.Next, separate the intestine from the internal organs, and stretch it out, taking care to obtain all of the intestine. Finally, use tweezers to roll the intestine on the filter paper to detach the adhesive tissue. Using this protocol, zebrafish of mixed sex were successfully intubated with different recombinant protein nanoparticles.Using the homemade oral intubation device, a low average percentage of mortality was achieved. To evaluate the delivery of the fluorescent nanoparticles inclusion bodies made with recombinant TNF alpha, a cytometry analysis was performed. The total amount of fluorescent cells was significantly higher in the nanoparticle-intubated group than in the control group both five and 24 hours after intubation.The intubation equipment is inexpensive, precise, and reusable. The whole intubation procedure can be done very quickly by one operator, and, importantly, the method is consistent between different operators. The combination of our intubation method with downstream analysis could be ideal for oral vaccine development.

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

Research

JoVE Journal

Immunology and Infection

7.7K visualizações.  Universitat Autònoma de Barcelona. Este método pode ajudar a responder a perguntas-chave no campo da vacinação da mucosa, como como a mucosa intestinal capta compostos biológicos e como tais biológicos modulam o sistema imunológico no local após a intubação. A principal vantagem dessa técnica é que é simples de configurar, rápido e mostra baixas taxas de mortalidade. Este método pode fornecer informações sobre a vacinação mucosa.Também pode ser aplicado a outros compostos, como produtos químicos, tó...