Name: the mouse isolated perfused kidney technique
Uploaded: 2016-11-17T14:00:00
Description: Watch this Scientific Journal Video about The Mouse Isolated Perfused Kidney Technique Video at JoVE.com

The authors would like to thank Hans-Joachim Schurek for invaluable scientific advice. The authors would like to thank Monique Carrel and Mich&#232;le Heidemeyer for excellent technical assistance, David Penton Ribas and Nourdine Faresse for a critical reading of the manuscript and Carsten Wagner and J&#252;rg Biber for the NaPi-2a antibody. This work was supported by the Swiss National Centre for Competence in Research "Kidney.CH" and by a project grant (310030_143929/1) from the Swiss National Science Foundation.

Alle procedurer, der involverer dyr, der er beskrevet i dette manuskript blev gennemført i henhold til schweizisk lovgivning og godkendt af den veterinære administration af kantonen Zürich, Schweiz. 1. Buffer Fremstilling <ol><li> Fremstille opløsninger 1 - 4 og antidiuretisk hormon (ADH) opløsning (tabel 1). </li><li> Forbered dialyse puffer (tabel 1). BEMÆRK: Dette er den anvendte buffer som dialysebufferen under perfusion. Senere vil erythr...

<ol>
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Musen isoleret perfunderede nyre er et værktøj til at studere nyrefunktion i et kontrolleret miljø ex vivo i 1 time, bygge bro mellem in vivo forsøg i intakte dyr, som kan være mangelfulde med virkningen af talrige systemiske faktorer, og in vitro eksperimenter i isolerede nephron segmenter eller dyrkede celler, som nødvendigvis forsømmer virkningen af ​​intakt organ struktur på funktion. Der er, til forfatternes viden, noget alternativ teknik med til at udføre denne specifikke op...

Den isolerede perfusion af organer har været genstand for en løbende indsats blandt fysiologer i mange årtier 1. Teknikken muliggør funktionen af ​​organet, uden systemiske påvirkninger, såsom blodtryk, hormoner, eller nerver, der skal undersøges. Carl Eduard Loebell anses for at være den første til at have beskrevet den succesfulde perfusion af en isoleret nyre, i 1849 2. Siden da har perfusionsapparatet undergået betydelig forfinelse. Frey og Gruber indført en kunstig lunge for iltning og pulserende pumper til kontinuerlig perfusion 2. Mens tidlige forskere hovedsageligt undersøgt nyrerne af store pattedyr-nemlig, svin 2 og hunde 3 -den første rapport af brugen af rotte nyrer, ved Weiss et al. , Var en milepæl i studiet af små pattedyr-orgel perfusion 4. Schurek et al. rapporterede nødvendigheden af ​​at tilføre mammale erytrocytter til perfusatet hvis tilstrækkelig renal tubulæriltning skulle opnås 5. Kritisk for langsigtede eksperimenter var indførelsen af kontinuerlig dialyse af bufferen af samme forskergruppe 6. I 2003 Schweda et al. var de første til at rapportere et isoleret funktionel mus perfunderet nyre (MIPK) 7, senere raffineres af Rahgozar et al. 18 og Lindell et al. 14. Mens teknisk mere udfordrende end rotten isoleret perfunderede nyre, brugen af ​​MIPK bærer den fordel, at anvendelsen af ​​en bred vifte af genetisk ændrede mus. Denne artikel præsenterer detaljerne i forfatternes metode til perfusion isolerede mus nyrer i 1 time. Metoden giver mulighed for løbende vurdering af renal flow, vaskulær modstand, hormon frigivelse, blodgas analyse, urinanalyse, og anvendelsen af ​​lægemidler. Ved at følge fremgangsmåden, kunne nyrer behandles til molekylær og biokemisk analyse, fastsættes for mikroskopi, ellertransplanteres i en recipient mus (figur 1). <img alt="figur 1" src="/files/ftp_upload/54712/54712fig1.jpg" /> Figur 1: Oversigt over mulige Input / Output til de isolerede perfunderede nyre. BGA: Blood gasanalyse. <a href="http://ecsource.jove.com/files/ftp_upload/54712/54712fig1large.jpg" target="_blank">Klik her for at se en større version af dette tal.</a> Denne teknik vil sandsynligvis modtage stigende opmærksomhed i de kommende år, da mange innovative applikationer drøftes med begyndelsen af langvarig normotermisk nyre perfusion før transplantation (med eller uden anvendelse af anti-afvisning eller genom-redigering narkotika) 8, 9, 10 , 11, den bioteknik af hele nyrer fra decellulariserede stilladser 12, og anvendelsen af høje doser af fluorescerende farvestoffer for multifoton billeddannelse 13 </sup&gt;. Det er også en ideel model, med hvilken at undersøge den rolle af specifikke gener under akut nyreskade 14. En trin-for-trin-protokollen er givet for at tillade andre laboratorier til at udføre isoleret mus nyre perfusion med succes. Først bliver sammensætningen og fremstillingen af ​​bufferen angivet. Derefter kirurgi beskrevet detaljeret og er vist de kritiske trin. For det tredje, data præsenteres som er repræsentative for en vellykket forberedelse: renal blodgennemstrømning, vaskulær modstand, glomerulære filtrationshastighed, og fraktioneret elektrolyt udskillelse-alle som funktionelle målinger af rentabilitet-og transmission elektron mikrografier af morfologi forskellige nephron segmenter af perfunderede nyrer fast efter 1 time af perfusion.

<table border="0" cellpadding="0" cellspacing="0" width="906">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Perfusion Circuit:</td>
			<td style="width:173px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Moist chamber 834/8</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-2901</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Cannular with basket and side port</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-2947</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Thermostat TC120-ST5&#160;</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-4544</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">ISM 827/230V Roller Pump Reglo Analogue</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-0114</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Reservoir jacketed for buffer solution 1L</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-3438</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Reservoir jacketed for buffer solution 0.5L</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-3436</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Pressure Transducer APT300&#160;</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-3862</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">TAM-D Plugsys Transducer</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-1793</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">SCP Plugsys servo controller</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-2806</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Windkessel&#160;</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-3717</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">HSE-USB data acquisition</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-3330</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Low-Flux Dialysator Diacap Polysulfone</td>
			<td style="width:173px;">B.Braun</td>
			<td align="right" style="width:119px;">7203525</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:447px;">PE-Tubing for aorta cannulation 1.19mm I.D. x 1.70mm O.D.</td>
			<td style="width:173px;">Scientific Commodities Inc.</td>
			<td style="width:119px;">BB31695-PE/8</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:447px;">Name</td>
			<td style="width:173px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Buffer reagents:</td>
			<td style="width:173px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Aminoplasmal 10%</td>
			<td style="width:173px;">B.Braun</td>
			<td align="right" style="width:119px;">134518064</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Sodium pyruvate</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">P2256-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">L-Glutamic acid monosodium salt hydrate</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">G1626-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">L-(-)-Malic acid sodium salt</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">M1125-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Sodium-L-Lactate</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">L7022-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">alpha-Ketoglutaric acid sodium salt</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">K1875-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">31434-1KG-R</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">NaHCO3</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">S5761-5KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">60130-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Urea</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">U5378-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Creatinine</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">C4255-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ampicillin</td>
			<td style="width:173px;">Roche</td>
			<td align="right" style="width:119px;">10835242001</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">MgCl2 * 6H2O</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">M2393-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">D-Glucose</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">G8270-1KG</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">CaCl2 * 6H2O&#160;</td>
			<td style="width:173px;">Riedel-de-Ha&#235;n</td>
			<td align="right" style="width:119px;">12074</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">NaH2PO4</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">S9638-500G</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">Na2HPO4</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">S0876-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Antidiuretic Hormone dDAVP</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">V2013-1MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">FITC-Inulin</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Filter used for erythrocyte filtration</td>
			<td>Macherey-Nagel</td>
			<td style="width:119px;">MN 615</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">BGA Analysis:</td>
			<td style="width:173px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">ABL 80 flex</td>
			<td style="width:173px;">Radiometer Medical ApS</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Electron Microscope:</td>
			<td style="width:173px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Philips CM100 TEM</td>
			<td style="width:173px;">FEI</td>
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the mouse isolated perfused kidney technique

Musen isoleret perfunderede nyre (MIPK) er en teknik til at holde en mus nyre under ex vivo betingelser perfunderes og funktionelle i 1 time. Dette er en forudsætning for at studere fysiologi af den isolerede organ og for mange innovative applikationer, der kan være muligt i fremtiden, herunder perfusion decellularization for nyre bioteknologi eller administration af anti-afvisning eller genom-redigering narkotika i høje doser til prime nyrerne til transplantation. I den tid af perfusionen, kan nyren manipuleres, nyrefunktionen kan vurderes, og forskellige lægemidler administreres. Efter proceduren, kan nyren transplanteres eller behandles med molekylær biologi, biokemisk analyse eller mikroskopi. Dette papir beskriver perfusatet og den kirurgiske teknik er nødvendig for ex vivo perfusion af mus nyrer. Nærmere oplysninger om perfusionsapparatet er givet, og data er præsenteret som viser viability i nyrerne forberedelse: renal blodgennemstrømning, vaskulær modstand, og urin data som funktionelle, transmission elektronmikrofotografier af forskellige nephron segmenter som morfologiske udlæsninger og western blots af transportproteiner i forskellige nephron segmenter som molekylær udlæsning.

Med beskrevet fremgangsmåden kan isolerede mus nyrer forblive levedygtige i mindst 1 time. Vi testede væv levedygtigheden efter 1 time med kontinuerlig perfusion med funktionelle (renal blodstrømning og vaskulær modstand, blodgasanalyse for venøs udstrømning, glomerulære filtrationshastighed, urin fraktioneret Na + og K + -udskillelse, og urinosmolalitet) og morfologiske (transmissionselektronmikroskopi mikroskopi, TEM) metoder i fire nyrer af vildtype C57BL ...

Watch this Scientific Journal Video about The Mouse Isolated Perfused Kidney Technique Video at JoVE.com

The Mouse Isolated Perfused Kidney Technique Video (Video) | JoVE

Musen isoleret perfunderede nyre (MIPK) er en teknik til at holde en mus nyre under ex vivo betingelser perfunderes og funktionelle i 1 time. Bufferne og kirurgisk teknik er beskrevet i detaljer.

Mus isoleret perfunderet Nyre Teknik

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 ...

Research

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Biology

The Mouse Isolated Perfused Kidney Technique

Chip-based Three-dimensional Cell Culture in Perfused Micro-bioreactors

Chip-based Three-dimensional Cell Culture in Perfused Micro-bioreactors Video (Video) | JoVE

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from ...

A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse 

A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse  Video (Video) | JoVE

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is pathologically characterized by extracellular deposition of &#946;-amyloid ...

Optical Mapping of Langendorff-perfused Rat Hearts

Optical Mapping of Langendorff-perfused Rat Hearts Video (Video) | JoVE

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer Video (Video) | JoVE

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney Video (Video) | JoVE

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

Characterization of the Isolated, Ventilated, and Instrumented Mouse Lung Perfused with Pulsatile Flow

Characterization of the Isolated, Ventilated, and Instrumented Mouse Lung Perfused with Pulsatile Flow Video (Video) | JoVE

The isolated, ventilated and instrumented mouse lung preparation allows steady and pulsatile pulmonary vascular pressure-flow relationships to be measured ...

Transplantation of Cells Directly into the Kidney of Adult Zebrafish

Transplantation of Cells Directly into the Kidney of Adult Zebrafish Video (Video) | JoVE

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic ...

Dissection of the Adult Zebrafish Kidney

Dissection of the Adult Zebrafish Kidney Video (Video) | JoVE

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

Generating Mice from Induced Pluripotent Stem Cells | Protocol (Video) | JoVE

Generering af mus afledt fra induceret pluripotente stamceller

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria Video (Video) | JoVE

Multi-parameter Måling af permeabilitetsovergang pore åbning i isolerede Mouse Heart Mitokondrier

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...