Name: simple and effective administration and visualization of microparticles in the circulatory system of small fishes using kidney injection
Uploaded: 2018-06-17T15:00:00
Description: Watch this Scientific Journal Video about Simple and Effective Administration and Visualization of Microparticles in the Circulatory System of Small Fishes Using Kidney Injection at JoVE.com

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).

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 ...

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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 (<strong class="...

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&#160;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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>SNARF-1-dextran, 70000 MW</td>
			<td>Thermo Fisher Scientific</td>
			<td>D3304</td>
			<td>Fluorescent probe. Any other appropriate polymer-bound fluorescent dye can be used as a microcapsule filler</td>
		</tr>
		<tr>
			<td>Albumin-fluorescein isothiocyanate conjugate (FITC-BSA)</td>
			<td>SIGMA</td>
			<td>A9771</td>
			<td>Fluorescent probe</td>
		</tr>
		<tr>
			<td>Rhodamine B isothiocyanate-Dextran (RITC-dextran)</td>
			<td>SIGMA</td>
			<td>R9379</td>
			<td>Fluorescent probe</td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>SIGMA</td>
			<td>C1016</td>
			<td>CaCO3 templates formation</td>
		</tr>
		<tr>
			<td>Sodium carbonate</td>
			<td>SIGMA</td>
			<td>S7795</td>
			<td>CaCO3 templates formation</td>
		</tr>
		<tr>
			<td>Poly(allylamine hydrochloride), MW 50000 (PAH)</td>
			<td>SIGMA</td>
			<td>283215</td>
			<td>Cationic polymer</td>
		</tr>
		<tr>
			<td>Poly(sodium 4-styrenesulfonate), MW 70000 (PSS)</td>
			<td>SIGMA</td>
			<td>243051</td>
			<td>Anionic polymer</td>
		</tr>
		<tr>
			<td>Poly-L-lysine [20 kDa] grafted with polyethylene glycol [5 kDa], g = 3.0 to 4.5 (PLL-g-PEG)</td>
			<td>SuSoS</td>
			<td>PLL(20)-g[3.5]-PEG(5)</td>
			<td>Final polymer to increase the biocompatibility of microcapsules</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>SIGMA</td>
			<td>S8776</td>
			<td>To dissolve applied polymers</td>
		</tr>
		<tr>
			<td>Water Purification System</td>
			<td>Millipore</td>
			<td>SIMSV0000</td>
			<td>To prepare deionized water</td>
		</tr>
		<tr>
			<td>Magnetic stirrer</td>
			<td>Stegler</td>
			<td></td>
			<td>For CaCO3 templates formation</td>
		</tr>
		<tr>
			<td>Eppendorf&#160;Research&#160;plus pipette, 1000 &#181;L</td>
			<td>Eppendorf</td>
			<td></td>
			<td>Dosing solutions</td>
		</tr>
		<tr>
			<td>Eppendorf&#160;Research&#160;plus pipette, 10 &#181;L</td>
			<td>Eppendorf</td>
			<td></td>
			<td>Dosing solutions</td>
		</tr>
		<tr>
			<td>Pipette tips, volume range 200 to 1000 &#181;L</td>
			<td>F.L. Medical</td>
			<td>28093</td>
			<td>Dosing solutions</td>
		</tr>
		<tr>
			<td>Pipette tips, volume range 0.1-10&#160;&#956;L</td>
			<td>Eppendorf</td>
			<td>Z640069</td>
			<td>Dosing solutions</td>
		</tr>
		<tr>
			<td>Mini-centrifuge Microspin 12, High-speed</td>
			<td>BioSan</td>
			<td></td>
			<td>For microcapsule centrifugation-washing procedure</td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes, 2 mL</td>
			<td>Eppendorf</td>
			<td>Z666513</td>
			<td>Microcapsule synthesis and storage</td>
		</tr>
		<tr>
			<td>Shaker Intelli-mixer RM-1L</td>
			<td>ELMY Ltd.</td>
			<td></td>
			<td>To reduce microcapsule aggregation</td>
		</tr>
		<tr>
			<td>Ultrasonic cleaner</td>
			<td></td>
			<td></td>
			<td>To reduce microcapsule aggregation</td>
		</tr>
		<tr>
			<td>Head phones&#160;</td>
			<td></td>
			<td></td>
			<td>To protect ears from ultrasound</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid</td>
			<td>SIGMA</td>
			<td>EDS</td>
			<td>To dissolve the CaCO3 templates</td>
		</tr>
		<tr>
			<td>Monosodium phosphate</td>
			<td>SIGMA</td>
			<td>S9638</td>
			<td>Preparation of pH buffers</td>
		</tr>
		<tr>
			<td>Disodium phosphate</td>
			<td>SIGMA</td>
			<td>S9390</td>
			<td>Preparation of pH buffers</td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>SIGMA</td>
			<td>S8045</td>
			<td>To adjust the pH of the EDTA solution and buffers</td>
		</tr>
		<tr>
			<td>Thermostat chamber</td>
			<td></td>
			<td></td>
			<td>To dry microcapsules on glass slide</td>
		</tr>
		<tr>
			<td>Hemocytometer blood cell count chamber</td>
			<td></td>
			<td></td>
			<td>To investigate the size distribution and concentration of the prepared microcapsules</td>
		</tr>
		<tr>
			<td>Fluorescent microscope Mikmed 2</td>
			<td>LOMO</td>
			<td></td>
			<td>In vivo visualization of microcapsules in fish blood</td>
		</tr>
		<tr>
			<td>Set of fluorescent filters for SNARF-1 (should be chosen depending on the microscope model; example is provided)</td>
			<td>Chroma</td>
			<td>79010</td>
			<td>Visualization of microcapsules with fluorescent probes</td>
		</tr>
		<tr>
			<td>Fiber spectrometer QE Pro</td>
			<td>Ocean Optics</td>
			<td></td>
			<td>Calibration of microcapsules under microscope</td>
		</tr>
		<tr>
			<td>Optical fiber QP400-2-VIS NIR, 400 &#956;m, 2 m</td>
			<td>Ocean Optics</td>
			<td></td>
			<td>To connect spectrometer with microscope port</td>
		</tr>
		<tr>
			<td>Collimator F280SMA-A</td>
			<td>Thorlabs</td>
			<td></td>
			<td>To connect spectrometer with microscope port</td>
		</tr>
		<tr>
			<td>Glass microscope slide</td>
			<td>Fisherbrand</td>
			<td>12-550-A3</td>
			<td>Calibration of microcapsules under microscope</td>
		</tr>
		<tr>
			<td>Coverslips, 22 x 22 mm</td>
			<td>Pearl</td>
			<td>MS-SLIDCV</td>
			<td>Calibration of microcapsules under microscope</td>
		</tr>
		<tr>
			<td>Glass microcapillaries Intra MARK, 10 &#181;L</td>
			<td>Blaubrand</td>
			<td>BR708709</td>
			<td>To collect fish blood</td>
		</tr>
		<tr>
			<td>Clove oil</td>
			<td>SIGMA</td>
			<td>C8392</td>
			<td>Fish anesthesia</td>
		</tr>
		<tr>
			<td>Lancet No 11</td>
			<td>Apexmed international B.V.</td>
			<td>P00588</td>
			<td>To cut the fish tail and release the steel needle from the tip of insulin autoinjector</td>
		</tr>
		<tr>
			<td>Heparin, 5000 U/mL</td>
			<td>Calbiochem</td>
			<td>L6510-BC</td>
			<td>For treating all surfaces that come in contact with fish blood during fish blood collection</td>
		</tr>
		<tr>
			<td>Seven 2 Go Pro pH-meter with a microelectrode</td>
			<td>Mettler Toledo</td>
			<td></td>
			<td>To determine fish blood pH</td>
		</tr>
		<tr>
			<td>Insulin pen needles Micro-Fine Plus, 0.25 x 5 mm</td>
			<td>Becton, Dickinson and Company</td>
			<td></td>
			<td>For injection procedure. Any thin needle (&#216; 0.33 mm or less) is appropriate</td>
		</tr>
		<tr>
			<td>Glass capillaries, 1 x 75 mm</td>
			<td>Hirschmann Laborger&#228;te GmbH &#38; Co</td>
			<td>9201075</td>
			<td>For injection procedure</td>
		</tr>
		<tr>
			<td>Gas torch</td>
			<td></td>
			<td></td>
			<td>To solder steel needle to glass capillary</td>
		</tr>
		<tr>
			<td>Microinjector IM-9B</td>
			<td>NARISHIGE</td>
			<td></td>
			<td>For precise dosing of microcapsules suspension</td>
		</tr>
		<tr>
			<td>Petri dishes, 60 mm x 15 mm, polystyrene</td>
			<td>SIGMA</td>
			<td>P5481</td>
			<td>For manipulations with fish under anesthesia</td>
		</tr>
		<tr>
			<td>Plastic spoon</td>
			<td></td>
			<td></td>
			<td>For manipulations with fish under anesthesia</td>
		</tr>
		<tr>
			<td>Damp sponge</td>
			<td></td>
			<td></td>
			<td>For manipulations with fish under anesthesia</td>
		</tr>
		<tr>
			<td>Dissection scissors</td>
			<td>Thermo Scientific</td>
			<td>31212</td>
			<td>To remove the gill cover from the fish head</td>
		</tr>
		<tr>
			<td>Pasteur pipette, 3.5 mL</td>
			<td>BRAND</td>
			<td>Z331767</td>
			<td>To moisten fish gills</td>
		</tr>
	</tbody>
</table>

simple and effective administration and visualization of microparticles in the circulatory system of small fishes using kidney injection

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 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&#160;2&#160;and 3) and, finally, the in vivo spectral recording of SNARF-1 fluorescence to monitor blood pH leve...

Watch this Scientific Journal Video about Simple and Effective Administration and Visualization of Microparticles in the Circulatory System of Small Fishes Using Kidney Injection at JoVE.com

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

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, ...

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Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

The Mouse Isolated Perfused Kidney Technique

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a ...

Hydrodynamic Renal Pelvis Injection for Non-viral Expression of Proteins in the Kidney

Hydrodynamic injection creates a local, high-pressure environment to transfect various tissues with plasmid DNA and other substances. Hydrodynamic tail ...