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Simple and Effective Administration and Visualization of Microparticles in the Circulatory System of Small Fishes Using Kidney Injection

Biology

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Summary

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.

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Borvinskaya, E., Gurkov, A., Shchapova, E., Karnaukhov, D., Sadovoy, A., Meglinski, I., Timofeyev, M. Simple and Effective Administration and Visualization of Microparticles in the Circulatory System of Small Fishes Using Kidney Injection. J. Vis. Exp. (136), e57491, doi:10.3791/57491 (2018).

Abstract

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.

Introduction

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.

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Protocol

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.

  1. To synthesize porous CaCO3 microcores enclosing the fluorescent dye, mix 2 mL of the SNARF-1-dextran solution (most polymer-bound fluorescent dye such as FITC-BSA can be used) at a concentration of ~2 mg/mL with 0.6 mL each of 1 mol/L solutions of CaCl2 and Na2CO3 under fast stirring.
    NOTE: Pay attention to the different sensitivities of fluorescent dyes to photobleaching; if a light-sensitive fluorescent probe (like SNARF-1) is used, the manipulation and storage of the microparticles must be performed with as little light as possible.
  2. After 5-10 s of agitation, transfer the suspension to 2 mL microcentrifuge tubes and centrifuge for 15 s at 10,000-12,000 g to pellet CaCO3 microcores.
  3. Discard the supernatant, wash the cores with ~2 mL of deionized water, and resuspend the pellet by shaking.
  4. Repeat the centrifugation-washing procedure three times in total. After the last centrifugation, discard the supernatant.
  5. Incubate the microcores for 1 min in an ultrasonic bath to reduce their aggregation.
    CAUTION: Do not forget to protect ears with headphones.
  6. To deposit the first polymeric layer on the templates, resuspend the cores in ~2 mL of a 4 mg/mL solution of poly(allylamine hydrochloride) (PAH) in 1 mol/L NaCl.
    1. Keep the microcores in the solution for ~5 min with constant shaking.
    2. After 15 s of centrifugation, discard the supernatant with the unbound PAH. Wash the covered microcores with deionized water at least 3 times through multiple centrifugation and washing steps. After the last centrifugation, discard the supernatant.
    3. Incubate the microcores for 1 min in an ultrasonic bath to reduce their aggregation.
      NOTE: If the applied fluorescent dye is cationic, start from poly(sodium 4-styrenesulfonate) (PSS) in 1 mol/L NaCl (see step 1.7).
  7. Repeat step 1.6 with ~2 mL of a 4 mg/mL solution of PSS (also containing 1 mol/L NaCl) to deposit the second polymeric layer on the templates.
  8. Repeat steps 1.6 and 1.7 six times to deposit 12 polymeric layers.
    NOTE: It is not recommended to take a long break (~12 h or more) in the procedure until ~3-5 layers have been deposited because CaCO3 microcores without coverage may tend to recrystallize. Note that PSS coverage causes a higher aggregation of the microcores, and the long pause is advisable only when PAH or poly-L-lysine grafted with polyethylene glycol (PLL-g-PEG) is the outmost layer.
  9. Incubate the covered microcores in 2 mg/mL PLL-g-PEG (~1 mL per microtube) for at least 2 h.
    1. Wash the microcores with water via sequential centrifugation and resuspension steps. After the last centrifugation, discard the supernatant.
  10. To obtain hollow microcapsules, dissolve the CaCO3 templates by adding 2 mL of 0.1 mol/L ethylenediaminetetraacetic acid (EDTA) solution (adjusted to pH 7.1 with NaOH) to the covered microcores.
    1. After ~5 min of incubation, centrifuge the microcapsules for 45 s and discard the supernatant with the EDTA.
    2. Repeat steps 1.10-1.10.1 twice.
  11. Wash the microcapsules with 0.9% NaCl three times through multiple centrifugation steps within 45 s followed by washing steps. After the last centrifugation step, discard the supernatant.
    NOTE: The final microcapsule solution for injection must be kept sterile (for example by adding ampicillin, 0.1 mg/mL), and the media should be biocompatible with the object of investigation (isotonic media with neutral pH).
  12. Estimate the concentration of the prepared microcapsules in a hemocytometer under a fluorescence microscope. Take a series of pictures of the microcapsules, measure the diameter of about a hundred microcapsules using ImageJ9 or equivalent software, and investigate the size distribution using a histogram.
  13. Store the obtained encapsulated probe in the dark.
    NOTE: After several washings in sterile 0.9% NaCl, the microcapsules can be stored for months at 4 °C. Complete drying of the microcapsules during storage is not recommended.

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.

  1. Place the required set of fluorescence filters to the fluorescent microscope according to the characteristics of the applied fluorescent dye and turn on the fluorescent lamp.
    1. Pull out the lever to the eyepieces.
      CAUTION: Excess light can damage the spectrometer matrix. Thus, make sure that the lever is in the "eyepiece" mode when the spectrometer is not used.
    2. Connect one end of the optical fiber to the spectrometer and the other end to a collimator. Using adapters, place the collimator in the focus of the camera tube or other available port of the fluorescent microscope.
    3. Turn on the spectrometer. Run the spectrometer control program and prepare the spectrometer for measurements.
  2. For calibration of the microcapsule batch, place ~5 µL of the microcapsule suspension (~10 000 microcapsules per µL in deionized water) on a microscope slide, and dry the drop in a dark place (for example, in a thermostat at 35 °C).
    1. To calibrate the spectral characteristics of the microencapsulated SNARF-1, use a series of buffers with different pH values in the range ~6-9. Drop ~10 µL of a buffer onto the dried microcapsules with SNARF-1-dextran and cover it with a coverslip.
    2. Place the glass slide on the microscope stage. Locate the microcapsules using a ×40 objective.
    3. Turn the microscope lever to the camera port. Register their fluorescence with the spectrometer.Turn the lever back to the eyepieces.
      NOTE: Make sure the spectral signal is far beyond the background level, and ensure that the microcapsules in the field of view are not in a bubble (by switching to a lower magnification if necessary). Avoid prolonged illumination of the same microcapsules. SNARF-1 is sensitive to photobleaching.
    4. Repeat step 2.2.3 for different microcapsules 10-15 times.
  3. Calculate the fluorescence peak ratios (for example, using R or Scilab) for all registered spectra and determine the regression line between the median ratio (for each buffer) and medium pH using the following formula:
    Equation 1
    NOTE: SNARF-1 has a spectrum with two peaks corresponding to the emission of the protonated and deprotonated dye, and the ratio between the peaks is responsive to the pH of the medium. In this study, the ratio between the fluorescence intensity for 605 and 660 nm is used. These wavelengths are chosen depending on the filter set used. a and b are coefficients to be determined by non-linear regression (for example, using R). Values 0.15 and 1.1 are, respectively, the minimum and maximum values of I605/I660 observed during the calibration.
  4. Collect about 10 µL of fish blood from approximately 5 adult animals. Place fish into a Petri dish with 1 µL/mL water suspension of clove oil for anesthesia and wait until the animal turns on its side and stops responding to pinches of the fin (usually ~2-3 min). Transfer the fish on a glass slide. Cut off the fish tail with a lancet and collect approximately 2 µL of fish blood from the tail vein.
    NOTE: To prevent blood clotting, treat the incision with heparin (5000 U/mL) and use heparinized glass capillaries and microcentrifuge tubes to collect the blood.
    1. Drip about 10 µL of blood with a pipette onto the tip of the microelectrode and determine the pH using a pH-meter.
    2. Drop the blood onto a slide with dried microcapsules and register the ratio of the fluorescence intensity as described for the calibration buffers (steps 2.2 - 2.3).
  5. Adjust the linear coefficient of the calibration curve to make the curve match the measurements in the fish blood (for more details see Borvinskaya et al. 20175).

3. Preparation for Injection

  1. Release the steel needle from the tip of the insulin pen (or syringe) by removing the plastic with a sharp lancet.
    NOTE: Any thin needle (Ø0.33 mm or less) or glass capillary (usually Ø1 mm) can be prepared for microinjection10,11.
  2. Insert the needle halfway into the glass microcapillary; quickly and gently solder it using a gas torch.
  3. Connect the glass microcapillary to the microinjector and flush it with sterile water three times. Ensure that the liquid flows through the needle.
  4. Fill the system with distilled water.
    NOTE: Make sure there are no bubbles in the system.

4. Injection

  1. Resuspend the prepared suspension of microcapsules (in sterile 0.9% NaCl, or any other media used for the injections, with a concentration of 0.5 to 6 million microcapsules per microliter) using the ultrasonic bath for 1 min.
    NOTE: Since the microcapsules tend to precipitate, during the following injection, shake the vial with the microcapsules mechanically (using a rotor) or manually every few minutes to resuspend them and prevent their aggregation.
  2. Place the fish into a Petri dish with anesthetic (0.1 mL/L of clove oil suspended in water) for ~2-3 min. Wait until the fish turns on its side and stops responding to a light pinch of the fin.
  3. Using a spoon, transfer the fish out of the anesthetic and gently place it on a damp sponge in a lateral position with the head towards the left (for right-handed person) or towards the right (for left-handed person).
  4. Just before the injection, suck 1-2 mm of air into the glass capillary connected with microinjector. Then, load it with approximately 2 µL of the dispersed microcapsules.
    NOTE: Before the injection, the microcapsule solution must be adjusted to the temperature at which the fish are kept.
  5. Gently stabilize the body of the fish on the sponge with non-dominant hand.
    1. Find the lateral line of the fish. Mentally select a segment that extends from the operculum to the end of the abdominal cavity. Find the middle of this segment. Put the needle 1 mm lower in the ventral direction.
    2. With a scraping movement, gently move the fish scales aside, and make a puncture. Insert the needle into the body at an angle of 45° to the table surface.
    3. Push the needle toward the spine until it carefully rests against it.
    4. Release approximately 1 µL of the microcapsules' suspension into the kidney, and slowly withdraw the needle.
      NOTE: To find the proper puncture site more easily, it is useful to practice finding the trunk kidney by transilluminating the fishes using a bottom light, as shown in Figure 2A and 2B.
  6. Rinse the fish from head to tail with a stream of water to remove any spilled microcapsules at the injection site.

5. In Vivo Visualization

  1. Use the dissection scissors to remove the gill cover from the fish head and denude the fish gills. Rinse the gills with water.
  2. Using a spoon, transfer the fish to a microscope slide, and place it on the stage of the fluorescent microscope.
    NOTE: Make sure that the gills of the fish do not dry out during successive procedures. To avoid this, periodically moisten them with water using a Pasteur pipette (approximately every 1-2 min).
  3. Darken the room and using low magnification (x10 objective) inspect the gills to find the fluorescent microcapsules.
    NOTE: When the procedure is used for introduction of some fluorescent particles into fish circulatory system, it is recommended to inspect gills of several individuals for unexpected fluorescent particles before injections. Gills of wild-type zebrafish do not have autofluorescence, but in some cases sporadic fluorescent particles (like food pieces or unicellular symbionts) may be present on gills. If necessary, such particles can be recognized based on their specific shape (for example, food pieces have irregular shape, unlike spherical microcapsules) or fluorescence spectrum (i.e., color).
    1. Switch the lens to a higher magnitude (×40 objective), and position a microcapsule or a group of microcapsules in the center of the field of view.
    2. Turn the lever to the port with a connected spectrometer. Record the spectral signal.
    3. Turn the lever back to the eyepiece.
    4. Repeat the measurements for different microcapsules several times.
  4. Transfer the fish to the aquarium with proper aeration for recovery.
    NOTE: With minimal practice, it is possible to perform the injection and signal recording at an approximate rate of 2-3 min per fish. The measurement can be repeated for one individual several times with the use of repeated low, harmless doses of anesthesia or another method of fixation. For long-term observation, use a system with continuous anesthesia12.

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Representative Results

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
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
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
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
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.

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Discussion

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.

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Disclosures

The authors have nothing to disclose.

Acknowledgements

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).

Materials

Name Company Catalog Number Comments
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

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References

  1. Rivas-Aravena, A., Sandino, A. M., Spencer, E. Nanoparticles and microparticles of polymers and polysaccharides to administer fish vaccines. Biol. Res. 46, (4), 407-419 (2013).
  2. Yashchenok, A. M., Jose, J., Trochet, P., Sukhorukov, G. B., Gorin, D. A. Multifunctional polyelectrolyte microcapsules as a contrast agent for photoacoustic imaging in blood. J. Biophotonics. 9, (8), 792-799 (2016).
  3. Pugach, E. K., Li, P., White, R., Zon, L. Retro-orbital injection in adult zebrafish. J. Vis. Exp. (34), e1645 (2009).
  4. Gurkov, A., Shchapova, Е, Bedulina, D., Baduev, B., Borvinskaya, E., Timofeyev, M. Remote in vivo stress assessment of aquatic animals with microencapsulated biomarkers for environmental monitoring. Sci. Rep. 6, e36427 (2016).
  5. Borvinskaya, E., Gurkov, A., Shchapova, E., Baduev, B., Shatilina, Z., Sadovoy, A., et al. Parallel in vivo monitoring of pH in gill capillaries and muscles of fishes using microencapsulated biomarkers. Biol. Open. 6, (5), 673-677 (2017).
  6. Diep, C. Q., Davidson, A. J. Transplantation of cells directly into the kidney of adult zebrafish. J. Vis. Exp. (51), e2725 (2011).
  7. Kreft, O., Javier, A. M., Sukhorukov, G. B., Parak, W. J. Polymer microcapsules as mobile local pH-sensors. J. Mater. Chem. 17, (42), 4471-4476 (2007).
  8. Sadovoy, A., Teh, C., Korzh, V., Escobar, M., Meglinski, I. Microencapsulated bio-markers for assessment of stress conditions in aquatic organisms in vivo. Laser Phys. Lett. 9, (7), 542-546 (2012).
  9. Ferreira, T., Rasband, W. S. ImageJ User Guide - Version 1.44. imagej.nih.gov/ij/docs/guide/ (2012).
  10. Poland, R. S., Bull, C., Syed, W. A., Bowers, M. S. Rodent brain microinjection to study molecular substrates of motivated behavior. J. Vis. Exp. (103), e53018 (2015).
  11. Liu, L., Duff, K. A technique for serial collection of cerebrospinal fluid from the cisterna magna in mouse. J. Vis. Exp. (21), e960 (2008).
  12. Johnston, L., Ball, R. E., Acuff, S., Gaudet, J., Sornborger, A., Lauderdale, J. D. Electrophysiological recording in the brain of intact adult zebrafish. J. Vis. Exp. (81), e51065 (2013).
  13. Gerlach, G. F., Schrader, L. N., Wingert, R. A. Dissection of the adult zebrafish kidney. J. Vis. Exp. (54), e2839 (2011).
  14. McKee, R. A., Wingert, R. A. Zebrafish renal pathology: Emerging models of acute kidney injury. Curr Pathobiol Rep. 3, (2), 171-181 (2015).
  15. Donath, E., Sukhorukov, G. B., Caruso, F., Davi, S. A., Möhwald, H. Novel hollow polymer shells by colloid-templated assembly of polyelectrolytes. Angew. Chem. Int. Ed. 37, (17), 2201-2205 (1998).
  16. Antipov, A. A., Shchukin, D., Fedutik, Y., Petrov, A. I., Sukhorukov, G. B., Möhwald, H. Carbonate microparticles for hollow polyelectrolyte capsules fabrication. Colloids Surf. A. 224, 175-183 (2003).
  17. Gaponik, N., Radtchenko, I. L., Gerstenberger, M. R., Fedutik, Y. A., Sukhorukov, G. B., Rogach, A. L. Labeling of biocompatible polymer microcapsules with near-infrared emitting nanocrystals. Nano Lett. 3, (3), 369-372 (2003).
  18. Volodkin, D. V., Larionova, N. I., Sukhorukov, G. B. Protein encapsulation via porous CaCO3 microparticles templating. Biomacromolecules. 5, (5), 1962-1972 (2004).
  19. Tzaneva, V., Perry, S. F. A Time differential staining technique coupled with full bilateral gill denervation to study ionocytes in fish. J. Vis. Exp. (97), e52548 (2015).

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