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.
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.
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 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.
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
2. Solutions Required
3. Preparing the Fluorescent Nanoparticle Suspension
4. Zebrafish Anesthetization and Oral Intubation
5. Zebrafish Intestine Dissection
6. Preparing Intestinal Cells for Cytometry
7. Preparing Intestine Cryosections for Confocal Microscopy
8. Preparing the Intestine for Real Time qPCR (RT-qPCR)
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 was calculated within 24 h post intubation. The experiments were performed by two operators, R had less experience working with zebrafish oral intubation than J. The results showed that even a new operator could independently perform the oral intubation experiment and easily achieve a high survival rate by this protocol. From our experience, the optimal fish size is 1 g, but we have successfully intubated fish as small as 0.5 g.
To better understand whether the fluorescent nanoparticles IBsTNFα (a recombinant cytokine protein nanostructured as inclusion bodies) were delivered to zebrafish by our method and taken up by the zebrafish intestine or not, we performed cytometry analysis. The nanoparticles (100 µg/fish, 50 µL) and PBS (control) were orally administered (by intubation) to zebrafish and the intestine was dissected at 5 h and 24 h post intubation. Total intestinal cells were prepared by step 6 and analyzed by detecting the fluorescence emission signal. The representative histograms of fluorescence intensity and the dot plots of fluorescent cell percentage are shown in Figure 2. The density of fluorescent cells is clearly higher in nanoparticle intubated group compared to the control group in both 5 h and 24 h (Figure 2A). The percentages of fluorescent cells are significantly higher in both 5 h (46.3%) and 24 h (43.0%) of nanoparticle intubated groups (Figure 2B).
To further study which part of the intestinal layer is involved in the nanoparticle uptake, we performed confocal microscopy analysis. The nanoparticles (20 µg/fish, 50 µL) and PBS (control) were orally intubated to zebrafish and the intestine was dissected at 5 h post intubation. The intestine sections were prepared by a frozen tissue method according to step 7 (Figure 3A). The confocal images of fluorescent nanoparticles in the intestine are shown in Figure 3B. The fluorescent nanoparticles were found in zebrafish intestine. We observed the fluorescence in the epithelial cells, lamina propria, and muscle cells.
To verify whether we could extract high quality RNA by our protocol, we analyzed the RNA extracted from the intestine with a bioanalyzer (also referred to as RNA analyzer here). Zebrafish were intubated orally with PBS (50 µL) or the nanoparticles (20 µg/fish, 50 µL). The intestines were dissected for RNA extraction at 24 h post intubation. We selected seven RNA samples to test with the analyzer. We found that all the tested RNA samples have high RNA integrity numbers ranging from 7.9 to 8.9 (Figure 4).
Figure 1: Oral intubation device. (A) Image of a 31 G Luer lock needle fixed on a 100 µL syringe with the silicon tube and pipette tip end covering the tip of the needle. (B) An enlarged image of the needle part. The black arrow indicates where the pipette tip end exceeds the tip of the needle. Please click here to view a larger version of this figure.
Figure 2: Flow cytometry analysis of fluorescent nanoparticles in zebrafish intestine via oral intubation. Zebrafish were treated with PBS or fluorescent IBsTNFα (100 µg) for 5 h and 24 h, respectively. (A) Representative histograms of fluorescence intensity. (B) Dot plots graph of fluorescent cells percentage. Each green dot represents the percentage of fluorescent cells in one individual, n ≥4. Data represent mean ± standard error of the mean (SEM). Differences were analyzed using one-way ANOVA. Significant differences with respect to control (**, p <0.01) Please click here to view a larger version of this figure.
Figure 3: Images of confocal microscopy analysis. Fish were orally intubated with PBS or 20 µg/fish fluorescent nanoparticles. The intestine was dissected at 5 h post intubation. (A) Zebrafish intestine embedded in OCT compounds. The intestine was placed with the natural orientation of a "Z" shape (a: anterior end; p: posterior end). (B) Confocal microscopy images of zebrafish intestine. The white arrows show that the fluorescent nanoparticles are taken up in the intestinal mucosa. Please click here to view a larger version of this figure.
Figure 4: RNA analyzer virtual gel image showing RNA extracted from 7 zebrafish intestines sampled post intubation. Sample number 1 and 2 are PBS intubated groups and sample number 3 to 7 are nanoparticle intubated groups. RNA integrity numbers (RIN) are given at the bottom ranging from 7.9 to 8.9. Please click here to view a larger version of this figure.
Operator | Volume | # fish intubated | Mean weight (g ± SD) |
# deaths | Mortality (%) |
R | 30 µL | 22 | 0.88 ± 0.14 | 3 | 13.6 |
R | 30 µL | 17 | 0.93 ± 0.19 | 0 | 0 |
J | 50 µL | 19 | 1.23 ± 0.31 | 1 | 5.2 |
J | 50 µL | 30 | 1.08 ± 0.40 | 2 | 6.6 |
Total | 88 | 1.03 ± 0.16 | 6 | 6.8 | |
SD: standard deviation of the mean |
Table 1: Comparison of zebrafish mortality caused by two operators using the protocol. The operator identification code, the intubated volume, the fish number, and the fish average weight in grams (g) are shown.
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 intubation experiment not only by observation of the animal for well-being (e.g., no bleeding) and for no leakage of the fluid administered, but also by checking the uptake of a bioactive nanoparticle in the intestine using downstream analysis (cytometry, confocal microscopy and qPCR). We show that intubated fluorescently labeled nanoparticles were found in the intestine.
On the practical side, the intubation equipment is cheap, precise and reusable: the basic device is made of a reusable 100 µL glass (e.g., Hamilton) syringe coupled to a 31 G needle with a piece of silicon on the top. A cut sterile tip is placed over the silicon tube and can be changed for each individual administration. The syringe is reusable, and the thin needle allows intubating small fish with a high possibility of success. Moreover, the cut sterile tip could be placed directly over the needle without the silicon tube which allows the intubation equipment easier to be made in a laboratory. The administered bioactive compound is clearly visible, and a correct administration can be monitored easily. A critical step during the intubation procedure is the needle entry. It is important that the needle is not tilted or inserted too much to avoid perforating the gills. The mortality observed with this method is very low (approximately 7%) and depends on the size of the fish. Gill perforation is the most common cause of fish death and when it happens fish die within the first hour. Although fish of 0.5 g can be easily intubated the optimal fish size is around 1 g. Another difference with the method developed by Collymore et al.is that the fish are fasted for 48 h to be sure that gastrointestinal tract is empty. The whole intubation procedure can be done very quickly (30 animals/h) by one operator and importantly, the method is consistent between different operators4. The intubation method is easy to learn and does not require much practice to master.
The intestine dissection procedure must be properly performed to obtain good quality samples for cytometry, confocal and qPCR analyses. The critical step at this point is the dissection of the entire intestine; the posterior section is fragile and easy to lose. Once the intestine is dissected, it can be further processed for cytometry (2 h protocol), qPCR analysis (2 h protocol until total RNA isolation) or confocal microscopy (1 h to have samples ready for cryosection). It is very important to keep track of the orientation of the intestine, especially for confocal microscopy. Cytometry analysis requires rapid processing, and the procedure cannot be stopped before completion. Whereas, RNA isolation and samples prepared for cryosection can be properly stored and processed at any time. For cytometry, good quality samples without debris clumping the machine can be isolated with this method and fast analysis for the presence of fluorescent cells can be performed easily on individuals. RNA quality monitoring showed that high quality RNA can be isolated from intestine allowing analysis of individual fish by qPCR. Finally, the cryosection preparation for confocal microscopy provides important structural information about the protein nanoparticle uptake. Our methods thus provide a model to test the dynamics of oral prophylactics or other compounds in the intestine.
The limitations of our study are the size of the fish since we did not test administration in fish smaller than 0.5 g and the use of a chemical anesthetic to sedate the animals. Some authors use cold water (0–4 °C) to anesthetize the zebrafish18 but in the context of animal welfare and European legal constraints we decided that MS-222 was the method of choice.
The zebrafish offers many advantages over other model systems including a complete set of gene data and the available transgenic lines. For immunologists, the transgenic lines (e.g., Tg mpx:GFP and Tg mpeg1:GFP) set a scene for the in vivo observation of immune cells such as macrophages and neutrophils19,20. Combining our oral intubation and downstream analysis with transgenic lines, could be ideal for identifying the cell types involving the uptake, transportation and processing of oral vaccines, nanoparticles and pathogens in fish.
The authors have nothing to disclose.
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 Ramón 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 Autònoma de Barcelona for helpful technical assistance.
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 |