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

Forfatterne bekrefter sterkt hjelp av Bogdan Osadchiy og Evgenii Protasov (Irkutsk State University, Russland) i utarbeidelsen av video protokollen. Denne forskningen ble støttet av den russiske Science Foundation (#15-14-10008) og russiske Foundation for grunnleggende forskning (#15-29-01003).

Alle eksperimentelle prosedyrer ble utført i samsvar med EU-direktivet 2010/63/EU for dyreforsøk og er godkjent av det dyr fag forskning komiteen av Institutt for biologi ved Irkutsk State University.
1. fabrikasjon av Microcapsules
Merk: Microcapsules bærer en fluorescerende farge tilberedes med en lag-på-lag-samling av motsatt ladet polyelectrolytes7,8. Alle prosedyrer ble utført ved romtemperatur.
...

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+renal+pathology:+Emerging+models+of+acute+kidney+injury.">Zebrafish renal pathology: Emerging models of acute kidney injury.</a> Curr Pathobiol Rep. 3 (2), 171-181 (2015).</li><li>Donath, E., Sukhorukov, G. B., Caruso, F., Davi, S. A., M&#246;hwald, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+hollow+polymer+shells+by+colloid-templated+assembly+of+polyelectrolytes.">Novel hollow polymer shells by colloid-templated assembly of polyelectrolytes.</a> Angew. Chem. Int. Ed. 37 (17), 2201-2205 (1998).</li><li>Antipov, A. A., Shchukin, D., Fedutik, Y., Petrov, A. I., Sukhorukov, G. B., M&#246;hwald, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbonate+microparticles+for+hollow+polyelectrolyte+capsules+fabrication.">Carbonate microparticles for hollow polyelectrolyte capsules fabrication.</a> Colloids Surf. A. 224, 175-183 (2003).</li><li>Gaponik, N., Radtchenko, I. L., Gerstenberger, M. R., Fedutik, Y. A., Sukhorukov, G. B., Rogach, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Labeling+of+biocompatible+polymer+microcapsules+with+near-infrared+emitting+nanocrystals.">Labeling of biocompatible polymer microcapsules with near-infrared emitting nanocrystals.</a> Nano Lett. 3 (3), 369-372 (2003).</li><li>Volodkin, D. V., Larionova, N. I., Sukhorukov, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+encapsulation+via+porous+CaCO3+microparticles+templating.">Protein encapsulation via porous CaCO3 microparticles templating.</a> Biomacromolecules. 5 (5), 1962-1972 (2004).</li><li>Tzaneva, V., Perry, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Time+differential+staining+technique+coupled+with+full+bilateral+gill+denervation+to+study+ionocytes+in+fish.">A Time differential staining technique coupled with full bilateral gill denervation to study ionocytes in fish.</a> J. Vis. Exp. (97), e52548 (2015).</li></ol>

For å demonstrere injeksjon av microparticles i sebrafisk nyre, brukte vi halvt gjennomtrengelig microcapsules lastet med en indikator fargestoff. Dermed inneholder protokollen instruksjoner for fabrikasjon av microcapsules ved hjelp av lag-på-lag montering av motsatt ladet polyelectrolytes7,8,15,16,17 ,18 (<strong class="...

Administrasjon av mikro-størrelse partikler i en dyr organisme er en viktig oppgave i slike områder som narkotika og vaksine levering1, blodkar visualisering2, transgene celle implantasjon3og liten optisk sensor implantasjon 4 , 5. men implantasjon prosedyren for Mikroskala partikler i vaskulære systemet av små forsøksdyr er vanskelig, spesielt for delikat vannlevende organismer. Det anbefales for populære forskning prøver som sebrafisk, som disse prosedyrene avklares med video-protokollene.
Intracardiac og kapillært microinjections krever opplært personale og unike mikrokirurgi fasiliteter for levering av microobjects til sebrafisk blod. Tidligere ble en retro-orbital manuell injeksjon3 foreslått som en enkel og effektiv metode for administrasjon av hele celler. Men i vår erfaring tar på grunn av det lite området av øyet capillary nettverket, det mye praksis for å oppnå ønsket resultat fra denne teknikken.
Her, beskriver vi en metode for robust og effektiv microparticle implantasjon i sirkulasjonssystemet ved manuell injeksjon direkte i nyre vev av voksen sebrafisk, som er rik på capillaries og nyre fartøy. Denne teknikken er basert på video protokollen for cellen transplantasjon i sebrafisk nyre6, men traumatisk og tidkrevende Mikrokirurgiske trinnene ble eliminert. Den foreslåtte metoden er preget av lav dødelighet (0-20%) og høy effektivitet (70-90% suksess), og det er lett å innføre bruk vanlig.
En viktig del av foreslåtte protokollen er visualisering av den implantert microparticles (hvis de er fluorescent eller fargelegges) i gill kapillærene, som gir mulighet for verifisering av injeksjon kvaliteten, en grov relative vurdering av antall injisert partikler, og oppdagelsen av spectral signalet for fysiologiske målinger direkte fra sirkulerende blod. Eksempel av de mulige anvendelser av beskrevet teknikken vi viser protokollen for i vivo målinger av sebrafisk blod pH bruker en microencapsulated fluorescerende sonde, SNARF-1, opprinnelig foreslått i 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

Systemisk administrasjonen av mikro-størrelse partikler i en levende organisme kan brukes for blodkar visualisering, narkotika og vaksine levering, implantasjon av transgene celler og små optiske sensorer. Imidlertid intravenøs microinjections til små dyr som er mest brukt i biologisk og veterinary laboratorier, er svært vanskelig og krever opplært personale. Her viser vi en robust og effektiv metode for innføring av microparticles i sirkulasjonssystemet av voksen sebrafisk (Danio rerio) ved injeksjon i fisk nyre. For å visualisere de introdusert microparticles i er blodkar, foreslår vi en enkel intravital tenkelig teknikk i fisk gjellene. I vivo overvåking av sebrafisk blod pH ble gjennomført med en injisert microencapsulated fluorescerende sondere, SNARF-1, å demonstrere en av de mulige anvendelser av beskrevet teknikken. Denne artikkelen gir en detaljert beskrivelse av innkapsling av pH-sensitive fargestoff og demonstrerer prinsippene for rask injeksjon og visualisering av de fått microcapsules for in vivo -opptak av fluorescerende. Den foreslåtte metoden av injeksjon er preget av en lav dødelighet (0-20%) og høy effektivitet (70-90% suksess), og det er lett å innføre bruk vanlig. Alle beskrevet prosedyrer kan utføres på andre små fiskearter, som guppies og medaka.

Innhentet resultatene kommer fra en av de tre hovedkategoriene presentert protokollen: dannelsen av fluorescerende microparticles av innkapsling av en fluorescerende farge (figur 1), nyre injeksjon av microcapsules med ytterligere effekt i Gill kapillærene (figur 2 og 3) og, til slutt, i vivo spectral opptak av SNARF-1 fluorescens overvåke blod pH-verdier (Figur 4...

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

Enkel og effektiv administrasjon og visualisering av Microparticles i sirkulasjonssystemet av små fisk med nyre injeksjon

Denne artikkelen viser prinsippene for en rask, minimal invasiv injeksjon av fluorescerende microparticles i circulatory system av små fisk og i vivo -visualisering av microparticles i fisk blod.

The systemic administration of micro-size particles into a living organism can be applied for vasculature visualization, drug and vaccine delivery, ...

Research

JoVE Journal

Biology

Computer-assisted Large-scale Visualization and Quantification of Pancreatic Islet Mass, Size Distribution and Architecture

The pancreatic islet is a unique micro-organ composed of several hormone secreting endocrine cells such as beta-cells (insulin), alpha-cells (glucagon), ...

Visualization of DNA Replication in the Vertebrate Model System DT40 using the DNA Fiber Technique

Visualisering av DNA Replication i Vertebrate Model System DT40 hjelp av DNA Fiber Technique

Maintenance of replication fork stability is of utmost importance for dividing cells to preserve viability and prevent disease. The processes involved not ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

Effektiv Utledning av Human Cardiac Forstadier og cardiomyocytes fra pluripotent humane embryonale stamceller med lite molekyl Induksjon

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

Visualisering av Caenorhabditis elegans Cuticle Structures Bruke Lipofile Vital Dye, DII

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

Visualisering og analyse av mRNA molekyler med fluorescens<em&gt; In Situ</em&gt; Hybridisering i<em&gt; Saccharomyces cerevisiae</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

En Face Detection av nitrogenoksid og Superoxide i endothelial lag av intakte Arterier

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular ...

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

Filtrering Isolering av nukleinsyrer: En enkel og 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

Musen isolerte perfuserte Nyre 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

Etter nyresvikt bekkenet injeksjon for ikke-viral uttrykk for proteiner i nyre

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

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

En enkel og effektiv metode i Vivo hjerte-spesifikke Gene manipulering av Intramyocardial injeksjon i mus

Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. ...