This article demonstrates the principles of a quick, minimally invasive injection of fluorescent microparticles into the circulatory system of small fishes and the in vivo visualization of the microparticles in fish blood.
The systemic administration of micro-size particles into a living organism can be applied for vasculature visualization, drug and vaccine delivery, implantation of transgenic cells and tiny optical sensors. However, intravenous microinjections into small animals, which are mostly used in biological and veterinary laboratories, are very difficult and require trained personnel. Herein, we demonstrate a robust and efficient method for the introduction of microparticles into the circulatory system of adult zebrafish (Danio rerio) by injection into the fish kidney. To visualize the introduced microparticles in the vasculature, we propose a simple intravital imaging technique in fish gills. In vivo monitoring of the zebrafish blood pH was accomplished using an injected microencapsulated fluorescent probe, SNARF-1, to demonstrate one of the possible applications of the described technique. This article provides a detailed description of the encapsulation of pH-sensitive dye and demonstrates the principles of the quick injection and visualization of the obtained microcapsules for in vivo recording of the fluorescent signal. The proposed method of injection is characterized by a low mortality rate (0-20%) and high efficiency (70-90% success), and it is easy to institute using commonly available equipment. All described procedures can be performed on other small fish species, such as guppies and medaka.
The administration of micro-size particles into an animal organism is an important task in such areas as drug and vaccine delivery1, vasculature visualization2, transgenic cell implantation3, and tiny optical sensor implantation4,5. However, the implantation procedure for microscale particles into the vascular system of small laboratory animals is difficult, especially for delicate aquatic organisms. For popular research specimens like zebrafish, it is advised that these procedures be clarified using video protocols.
Intracardiac and capillary microinjections require trained personnel and unique microsurgery facilities for the delivery of microobjects into zebrafish blood. Previously, a retro-orbital manual injection3 was suggested as an easy and effective method for the administration of whole cells. However, in our experience, because of the small area of the eye capillary network, it takes much practice to achieve the desired outcome from this technique.
Herein, we describe a method for robust and efficient microparticle implantation into the circulatory system by manual injection directly into the kidney tissue of adult zebrafish, which is rich in capillaries and renal vessels. This technique is based on the video protocol for cell transplantation into the zebrafish kidney6, but the traumatic and time-consuming microsurgical steps were eliminated. The proposed method is characterized by low mortality (0-20%) and high efficiency (70-90% success), and it is easy to institute using commonly available equipment.
An important part of the proposed protocol is the visualization of the implanted microparticles (if they are fluorescent or colorized) in the gill capillaries, which allows for the verification of the injection quality, a rough relative assessment of the number of injected particles, and the detection of the spectral signal for physiological measurements directly from the circulating blood. As an example of the possible applications of the described technique, we demonstrate the protocol for in vivo measurements of zebrafish blood pH using a microencapsulated fluorescent probe, SNARF-1, originally suggested in Borvinskaya et al. 20175.
All experimental procedures were conducted in accordance with the EU Directive 2010/63/EU for animal experiments and have been approved by the Animal Subjects Research Committee of Institute of Biology at Irkutsk State University.
1. Fabrication of Microcapsules
NOTE: Microcapsules carrying a fluorescent dye are prepared using a layer-by-layer assembly of oppositely charged polyelectrolytes7,8. All procedures were performed at room temperature.
2. Preparation of Optical Setup and Calibration of Microencapsulated SNARF-1
Note: Rough pH measurements with microencapsulated SNARF-1 can be made using images in two channels of a fluorescent microscope7, but in this protocol a one-channel fluorescent microscope connected to a fiber spectrometer was applied.
3. Preparation for Injection
4. Injection
5. In Vivo Visualization
The obtained results come from one of the three main categories of the presented protocol: the formation of fluorescent microparticles by encapsulation of a fluorescent dye (Figure 1), the kidney injection of microcapsules with further visualization in gill capillaries (Figure 2 and 3) and, finally, the in vivo spectral recording of SNARF-1 fluorescence to monitor blood pH levels (Figure 4).
The layer-by-layer approach using coating of the template CaCO3 cores enclosing the dye by multiple layers of oppositely charged polymers (PAH and PSS) and an outermost layer of a biocompatible polymer (PLL-g-PEG) is a simple and cost-effective method allowing to encapsulate different fluorescent probes such as SNARF-1-dextran, FITC-BSA or others (Figure 1A). As a result, hollow microcapsules of micrometric size with stable semi-permeable elastic shells and loaded with the fluorescent dye are obtained (Figure 1B). The microcapsules fabricated by this technique are typically non-uniform, with a normal distribution of particle size and characteristic median size in each batch (Figure 1С).
Figure 1: Layer-by-layer synthesis and characterization of hollow polyelectrolyte microcapsules loaded with a fluorescent dye. (A) Scheme of a microcapsules assembly (drawn from a similar scheme published in Gurkov et al. 20164). (B) Picture of hollow microcapsules loaded with fluorescent dye FITC-BSA. (C) Size characterization of the batch of microcapsules from Figure 1B. Please click here to view a larger version of this figure.
Robust and efficient microparticle implantation into the circulatory system of small fishes can be performed by manual injection directly into the fish kidney. The puncture in the proper site of the fish body (trunk kidney) is crucial for rapid delivery of microparticles by manual injection. The fish kidney is a hematopoietic organ and contains pigmented melanophores, and thus, it is well colored. Because it is nestled between transparent chambers of the swim bladder, it is easy to identify the organ in the intact animal with weak pigmentation or by transillumination of small fishes using a bottom light source, as shown in Figure 2A and 2B.
Figure 2: Localization of the proper site for kidney injection. (A) Trans-illumination of the zebrafish demonstrates the localization of the trunk kidney (arrow). (B) Scheme illustrates how to identify the proper puncture site. The white dotted line indicates the lateral line of the abdominal segment of the fish. The arrow indicates the site for puncture and the proper direction of injection toward the spine. The swim bladder is designated by the green line. (C) Visualization of zebrafish kidney following the removal of organs and the body wall. An arrow points to the central bulge of the trunk kidney. (D) A sagittal histological section of D. rerio illustrates the general anatomy of the internal organs of the adult fish (H&E stain). (E) Micrograph of a fish kidney (dotted) scaled from (D) with an arrow pointing to the lumen of the posterior cardinal vein. Asterisks indicate the swim bladder. Please click here to view a larger version of this figure.
To check the success of the injection procedure, a rapid visual inspection of the gills at low magnification (x10-20 objective) should be made after cutting the fish gill cover (Figure 3A). Despite most of the microcapsules remaining at the injection site or spilling into the body cavity (Figure 3B), if the injection is performed correctly, it is possible to observe the fluorescent microcapsules freely floating in the gill capillaries of the denuded fish gills immediately after the injection (Figure 3C). If no fluorescent particles are detected in the gills, it is possible to repeat the injection in the same puncture. Successful delivery to the bloodstream should be obtained in approximately 70-90% of the total injected fish.
Figure 3: Kidney injection of microcapsules with further visualization in gill capillaries. (A) The overall scheme of the microcapsule delivery into the zebrafish bloodstream. (B) Fluorescent image of a zebrafish after the injection of the microcapsules with green FITC-BSA dye (the puncture site is indicated by an arrow). (C) This picture of the gills of fish, scaled from (B), demonstrates the successful delivery of microcapsules by kidney injection (gills of wild-type zebrafish have no autofluorescence). Please click here to view a larger version of this figure.
During the injection directly into the fish trunk kidney, extensive bruising normally forms beneath the puncture site, indicating damage to the parenchyma capillaries or the renal vessels. There is no blood leakage out of the body because the puncture appears to quickly contract. Despite internal bleeding, individuals can still recover with approximately 80-90% surviving through the procedure (Table 1). It is also known that fish species can regenerate nephrons de novo after injury13,14. If more than 20% of fish are dying, one must ensure that the anesthetization is performed properly.
Encapsulated fluorescent dye | n | Concentration, microcapsules per µL | Injection volume, µL | Average diameter of microcapsules, µm | Successful delivery into the fish bloodstream, % | Mortality after injection, % | |
1 h | 1 day | ||||||
Injection into the trunk kidney | |||||||
FITC-BSA | 36 | 4 x 106 | 1.6 | 2.7 | 94 | 8 | 3 |
SNARF-1-dextran | 29 | 3-6 x 105 | 1-2 | 5.1 | 72 | 7 | 12 |
RITC-dextran | 20 | 6 x 106 | 2 | 2.0 | 70 | 0 | 0 |
0.9% NaCl | 41 | – | 1-2 | – | – | 5 | 0 |
Retro-orbital injection | |||||||
FITC-BSA | 9 | 1 x 105 | 2 | 4.2 | 11 | 22 | 0 |
RITC-dextran | 11 | 6 x 106 | 1 | 2.0 | 9 | 18 | 0 |
Table 1. Safety and efficiency of the delivery of microcapsules into the zebrafish bloodstream. The success of the procedure is determined by the presence of the fluorescent materials in the fish gill capillaries.
If the concentration of the injected microparticles is insufficient, too few particles may be available to be rapidly visualized in fish gills. At the same time, a suspension with a too high concentration can clog the needle. In case of layer-by-layer assembled microcapsules with median diameter ~2-5 µm, a concentration of approximately 4*105-6*106 microcapsules per µL is optimal for effortless detection in fish gills after injection into fish kidney (Table 2).
Concentration, microcapsules per µL | Number of microcapsules in a microscope field of view (×20 objective) in the gills of different individuals (n = 7 per concentration) | ||||||
4 x 106 | > 100 | > 100 | 0 | > 100 | ≈ 70 | ≈ 50 | > 100 |
4 x 105 | ≈ 10 | ≈ 20 | 0 | 0 | ≈ 20 | ≈ 40 | ≈ 15 |
4 x 104 | 0 | 3 | 0 | 0 | 0 | 0 | 4 |
4 x 103 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Table 2. Record of the visual counting of FITC-BSA-containing microcapsules in D. rerio gills after injection (1.6 µL) into the trunk kidney.
The proposed method of microparticles' implantation into fish circulatory system can be applied for in vivo monitoring of zebrafish blood pH by microencapsulated fluorescent probe, SNARF-1 (Figure 4). SNARF-1 has a spectrum with two peaks corresponding to the emission of the protonated and deprotonated dye (Figure 4A), thus the calibration curves of the ratio between the peaks at different pH of the media can be plotted (Figure 4B). The components of the blood influence the readout of the encapsulated SNARF-1 (Figure 4B), which can be evaluated experimentally by simultaneous measurement of blood pH by microcapsules and a pH-meter with a glass microelectrode. A putative calibration curve for microcapsules in zebrafish blood should be plotted by shifting the buffers' curve by the coefficient equal to the pH difference measured experimentally (see Borvinskaya et al. 20175 for details).
Blood pH in zebrafish gill capillaries remains stable during at least several hours after the injection of microcapsules (Figure 4C). At the same time, a short hypercapnic exposure leads to a statistically significant decrease of blood pH, which demonstrates applicability of the method for in vivo research on small fishes.
Figure 4: Representative example of monitoring of zebrafish blood рН by registration of the fluorescence spectra of microencapsulated dye SNARF-1. (A) Spectra of the prepared microcapsules loaded with SNARF-1 in sodium phosphate buffers at different pH. (B) Calibration curves of the prepared pH-sensitive microcapsules in sodium phosphate buffers and in extracted zebrafish blood. For all measurements, the mean ± standard deviation is depicted. (C) A representative example of the in vivo monitoring of zebrafish blood рН by encapsulated fluorescent SNARF-1 dye in zebrafish gill capillaries. In control conditions, blood pH remains stable during 4 hours after injection of microcapsules, while 5 minutes exposure under severe hypercapnia (900-1000 mg/L of dissolved CO2) causes acidification of the fish blood. Asterisk indicates statistically significant difference from the parallel control group with p < 0.01 (Mann-Whitney U test). Please click here to view a larger version of this figure.
To demonstrate the injection of microparticles into the zebrafish kidney, we used semi-permeable microcapsules loaded with an indicator dye. Thus, the protocol contains instructions for the fabrication of the microcapsules using the layer-by-layer assembly of oppositely charged polyelectrolytes7,8,15,16,17,18 (Figure 1A). An advantage of this technology is that it is easy to perform with available laboratory equipment. Depending on the conditions and compounds used, the fabricated microcapsules can range from 0.5 to 100 µm with nanometers-thick polyelectrolyte shell15. The synthesis parameters described in this manuscript result in elastic microcapsules consisting of 12 layers (in addition to the final biocompatible layer) of polymers approximately 2-6 µm in size (Figure 1B and 1C). The most important step determining the size of the microcapsules is the formation of the template microcores. This process involves the spontaneous formation of calcium carbonate crystals, and therefore, the obtained particles are non-uniform. Hence, a characterization of the microcapsule size distribution should be performed for every batch.
The important stage of this protocol is the optimization of the delivery of microparticles into the zebrafish circulatory system without the use of micromanipulation techniques. The administration of whole cells by retro-orbital injection has been previously described in detail3. However, in our experience, it is not easy for novice users to gain the injection efficiency described by the authors because the retro-orbital sinus is very small and located close to the pharynx and gill arches, which are easy to accidentally injure with a needle. Since less than a millimeter accuracy is required for the injections, the injecting properly is a rather difficult task. An equally quick and more efficient alternative (Table 1) is to inject directly into the fish kidney parenchyma. During the injection, the needle mechanically damages renal capillaries and blood vessels (e.g., the largest posterior cardinal vein), which allows entrance of microparticles into the circulatory system (Figure 2E). Finally, the central bulge of the trunk kidney in adult zebrafish is large enough (up to 2 mm) for a manual injection without microsurgical equipment.
The proper positioning of the injection needle is critical for a successful manual administration. You can practice finding the trunk kidney in intact animals by transilluminating fishes using a bottom light, as shown in Figure 2A and 2B. The scheme in Figure 2B demonstrates how to properly perform the injection. If the procedure fails to administer microparticles into the blood stream, re-injection can be made at the same puncture within a short time frame with virtually no effect on the survival rate of the individuals.
Injection into the trunk kidney of adult zebrafish (also successfully tested on adult guppy Poecilia reticulata) is an effective method of microparticle delivery into the fish bloodstream with an allowable animal mortality; however, it is not perfectly suitable for drug administration due to variations in the injected volume. A weak point of this technique is a significant leakage of the solution out of the kidney into the abdomen because of the rapid administration. Nevertheless, a relative amount of the substance injected into bloodstream can be roughly estimated using visualization in the gill capillaries (Table 2). There is no strict limitation of the injection volume, but the administration of more than 1 µL of the suspension appears to be ineffective. The finest glass capillaries can be applied for the microinjection; however, because a large number of microcapsules tends to incite aggregation, we recommend the use of 31-29G (Ø 0.33-0.25 mm) steel needles to avoid clogs in the needle lumen.
The success of microcapsule delivery through kidney injection into the bloodstream should be monitored using the rapid inspection for the presence of the fluorescent particles in the gills. Gills are easily accessible organs for in vivo observations. The gill filaments are a network of blood capillaries covered by a thin layer of respiratory epithelium, which makes intravital imaging of the blood flow in the gills particularly convenient (a kind of natural optical window). To perform the observation, the cartilage gill cover can be cut to expose the filaments. This procedure is safe for fish, which can live with denuded gills in well aerated water without a decrease in life expectancy. Moreover, the gills of large fish can be examined under a microscope simply by pushing the operculum out of the way19. In case of a successful injection, fluorescent microparticles immediately appear in the gills (Figure 3). Note that implanted microparticles are considerably dissolved in the circulating blood volume, and a reduced number of particles reach the gill capillaries, thus sufficient concentration of microparticles should be selected for detection in fish gills after injection into fish kidney (Table 2).
The proposed procedure for implantation can be applied in a wide range of studies involving different species of small fishes. Despite the technique having been developed and optimized for injection of fluorescent microparticles into circulatory system, it can also be applied for implantation of non-colored micro/nanoparticles or dissolved substances; however, in this case the effectiveness of injection should be verified in some other way. The currently described steps are optimal for such purposes as physiological monitoring using different optical micro- or nanosensors, visualization of vasculature, delivery of vaccines or drugs on some optically visible carriers, and implantation of genetically modified cells.
The authors have nothing to disclose.
Authors greatly acknowledge the help of Bogdan Osadchiy and Evgenii Protasov (Irkutsk State University, Russia) in preparation of the video protocol. This research was supported by the Russian Science Foundation (#15-14-10008) and the Russian Foundation for Basic Research (#15-29-01003).
SNARF-1-dextran, 70000 MW | Thermo Fisher Scientific | D3304 | Fluorescent probe. Any other appropriate polymer-bound fluorescent dye can be used as a microcapsule filler |
Albumin-fluorescein isothiocyanate conjugate (FITC-BSA) | SIGMA | A9771 | Fluorescent probe |
Rhodamine B isothiocyanate-Dextran (RITC-dextran) | SIGMA | R9379 | Fluorescent probe |
Calcium chloride | SIGMA | C1016 | CaCO3 templates formation |
Sodium carbonate | SIGMA | S7795 | CaCO3 templates formation |
Poly(allylamine hydrochloride), MW 50000 (PAH) | SIGMA | 283215 | Cationic polymer |
Poly(sodium 4-styrenesulfonate), MW 70000 (PSS) | SIGMA | 243051 | Anionic polymer |
Poly-L-lysine [20 kDa] grafted with polyethylene glycol [5 kDa], g = 3.0 to 4.5 (PLL-g-PEG) | SuSoS | PLL(20)-g[3.5]-PEG(5) | Final polymer to increase the biocompatibility of microcapsules |
Sodium chloride | SIGMA | S8776 | To dissolve applied polymers |
Water Purification System | Millipore | SIMSV0000 | To prepare deionized water |
Magnetic stirrer | Stegler | For CaCO3 templates formation | |
Eppendorf Research plus pipette, 1000 µL | Eppendorf | Dosing solutions | |
Eppendorf Research plus pipette, 10 µL | Eppendorf | Dosing solutions | |
Pipette tips, volume range 200 to 1000 µL | F.L. Medical | 28093 | Dosing solutions |
Pipette tips, volume range 0.1-10 μL | Eppendorf | Z640069 | Dosing solutions |
Mini-centrifuge Microspin 12, High-speed | BioSan | For microcapsule centrifugation-washing procedure | |
Microcentrifuge tubes, 2 mL | Eppendorf | Z666513 | Microcapsule synthesis and storage |
Shaker Intelli-mixer RM-1L | ELMY Ltd. | To reduce microcapsule aggregation | |
Ultrasonic cleaner | To reduce microcapsule aggregation | ||
Head phones | To protect ears from ultrasound | ||
Ethylenediaminetetraacetic acid | SIGMA | EDS | To dissolve the CaCO3 templates |
Monosodium phosphate | SIGMA | S9638 | Preparation of pH buffers |
Disodium phosphate | SIGMA | S9390 | Preparation of pH buffers |
Sodium hydroxide | SIGMA | S8045 | To adjust the pH of the EDTA solution and buffers |
Thermostat chamber | To dry microcapsules on glass slide | ||
Hemocytometer blood cell count chamber | To investigate the size distribution and concentration of the prepared microcapsules | ||
Fluorescent microscope Mikmed 2 | LOMO | In vivo visualization of microcapsules in fish blood | |
Set of fluorescent filters for SNARF-1 (should be chosen depending on the microscope model; example is provided) | Chroma | 79010 | Visualization of microcapsules with fluorescent probes |
Fiber spectrometer QE Pro | Ocean Optics | Calibration of microcapsules under microscope | |
Optical fiber QP400-2-VIS NIR, 400 μm, 2 m | Ocean Optics | To connect spectrometer with microscope port | |
Collimator F280SMA-A | Thorlabs | To connect spectrometer with microscope port | |
Glass microscope slide | Fisherbrand | 12-550-A3 | Calibration of microcapsules under microscope |
Coverslips, 22 x 22 mm | Pearl | MS-SLIDCV | Calibration of microcapsules under microscope |
Glass microcapillaries Intra MARK, 10 µL | Blaubrand | BR708709 | To collect fish blood |
Clove oil | SIGMA | C8392 | Fish anesthesia |
Lancet No 11 | Apexmed international B.V. | P00588 | To cut the fish tail and release the steel needle from the tip of insulin autoinjector |
Heparin, 5000 U/mL | Calbiochem | L6510-BC | For treating all surfaces that come in contact with fish blood during fish blood collection |
Seven 2 Go Pro pH-meter with a microelectrode | Mettler Toledo | To determine fish blood pH | |
Insulin pen needles Micro-Fine Plus, 0.25 x 5 mm | Becton, Dickinson and Company | For injection procedure. Any thin needle (Ø 0.33 mm or less) is appropriate | |
Glass capillaries, 1 x 75 mm | Hirschmann Laborgeräte GmbH & Co | 9201075 | For injection procedure |
Gas torch | To solder steel needle to glass capillary | ||
Microinjector IM-9B | NARISHIGE | For precise dosing of microcapsules suspension | |
Petri dishes, 60 mm x 15 mm, polystyrene | SIGMA | P5481 | For manipulations with fish under anesthesia |
Plastic spoon | For manipulations with fish under anesthesia | ||
Damp sponge | For manipulations with fish under anesthesia | ||
Dissection scissors | Thermo Scientific | 31212 | To remove the gill cover from the fish head |
Pasteur pipette, 3.5 mL | BRAND | Z331767 | To moisten fish gills |