Die Autoren möchten die NINDS, FightSMA und Familien von SMA für die finanzielle Unterstützung zu bestätigen. SEGL durch NINDS Bildungsprämie # 5T32NS077984-02 unterstützt.

Alle im Protokoll aufgeführten Verfahren genehmigt worden Institut für Tier Benutzung und Pflege Ausschuss (IACUC) der Ohio State University. 1. Herstellung von Workspace <ol><li> Gather nassem Eis, um die Maus Welpen zu betäuben, einen leeren Käfig, um den Damm aus dem Wurf, einem Binokular, einer Lichtquelle, die in einem Winkel zu der Injektion (Verwendung einer Lichtquelle mit einer 90 ° positioniert werden kann trennen &#160; Winkel zu der Injektionsstelle verdeckt die Vene), eine saubere Oberfläche, um das Tier für die Einspritzung, Wattestäbchen, 3/10 cc-Insulinspritze mit 3/8 &quot;30 G-Nadel (eines pro Tier) und 1% Evans Blue-Farbstoff ( mit Phosphat gepufferter Salzlösung (PBS)) Lösung für die Ausbildung gemacht. </li><li> Entfernen Sie die Staumauer in einen separaten Käfig, während die Manipulation der Welpen. </li></ol> 2. Injektionsverfahren <ol><li> Legen Sie eine einzelne Welpe direkt auf dem nassen Eis für 30-60 sec, um das Tier zu betäuben. Lassen Sie keine ter Tier auf Eis zu lange wegen einer Gefahr der Unterkühlung im Zusammenhang mit Komplikationen einschließlich Kammerflimmern, Gewebehypoxie und metabolische Azidose. HINWEIS: Unsere Erfahrung ist, dass 30 bis 60 sec ist ausreichend, um die Welpen Bewegungen verlangsamen, um die Injektion zu ermöglichen. Wenn tiefer Narkose erforderlich ist, kann Inhalationsmittel, wie 1-2% Isofluran geeignet. </li><li> Während das Tier auf dem Eis, laden Sie die Spritze mit 30 ul Evans blauen Farbstoff. </li><li> Wenn das Tier vollständig betäubt wird, durch den Mangel an Bewegung auf dem Eis, während noch atmete bestätigt, verschieben Sie sie unter dem Mikroskop. Für einen rechtshändigen Injektion gegen Schnauze des Tieres nach rechts. Platzieren Sie den linken Zeigefinger auf die Schnauze und dem linken Mittelfinger kaudal der Ohrhörer, so dass der Ohrhörer ist zwischen dem Zeige- und Mittelfinger (Abbildung 1). </li><li> Untersuchen nur der vordere Ohrstöpsel für eine oberflächliche Kapillare, wenn die Haut Stell bewegt. Diese Kapillare ist nicht das Ziel, aberIdentifikation ist wichtig für zeitliche Venenerkennung. Als nächstes suchen Sie einen dunklen, schattenhaften Venen schlechter als die Kapillare, die unabhängig von Haut Position fixiert bleibt. Die zeitliche Vene erscheint schemen läuft dorsal nach ventral und mündet in die Halsschlagader (Abbildung 1). </li><li> Geben Sie den zeitlichen Vene mit der Nadel Kegel up. Wenn richtig eingesetzt, ist es möglich, sehen die Nadelkegel mit Blut zu füllen durch die Haut. Dann den Kolben langsam drücken und Mitteilungsblanc der Vene an der Seite des Gesichts. </li><li> Dass die Nadel in der Vene für eine zusätzliche 10-15 sec, um eine Rückströmung der Einspritzmittel zu verhindern bleibt. </li></ol> 3. Post-Injektion <ol><li> Nach einer korrekten Injektion sollte der Welpe blau fast sofort einzuschalten. Entfernen Sie die Nadel und benutzen sanfter Gewalt zu einem Wattestäbchen auf die Injektionsstelle, bis die Blutgerinnsel gelten. </li><li> Überwachen Sie den Welpen für Anzeichen von Leiden. Lassen Sie den Welpen 2-3 min zu erholen und aufwärmen, recognize wenn der Welpe bei Bewusstsein ist, aufrecht und bewegt, vor der Rückkehr in den Käfig. Cup die Welpen in behandschuhten Händen des Untersuchers, angemessene Wärme in der Erholung bei Bedarf Hilfe zu leisten. Alternativ legen Sie ein Heizkissen unter dem Käfig zu erleichtern Erwärmung des eingespritzten pup. In der Regel nach einem Evans blauen Farbstoff Injektion, einschläfern Welpen. Enthalten Farbstoff nicht bei der Injektion Testmaterial. </li><li> Platzieren Sie den Welpen wieder in den Heimkäfig und sicherstellen, der Welpe ist mit Bettwäsche und / oder nestlet beschichtet, um Wiederaufnahme durch den Damm zu sichern. </li><li> Verwenden Sie eine neue Spritze und Wattestäbchen für jeden Welpen, um die Sterilität zu erhalten. </li></ol>

<ol>
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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+neonatal+intravascular+injections:+modeling+newborn+disease.">Murine neonatal intravascular injections: modeling newborn disease.</a> Journal of the American Association for Laboratory Animal Science : JAALAS. 46, 50-54 (2007).</li><li>Glascock, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+of+therapeutic+agents+through+intracerebroventricular+(ICV)+and+intravenous+(IV)+injection+in+mice.">Delivery of therapeutic agents through intracerebroventricular (ICV) and intravenous (IV) injection in mice.</a> J. vis. Exp. (56), (2011).</li><li>Sands, M. S., Barker, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Percutaneous+intravenous+injection+in+neonatal+mice.">Percutaneous intravenous injection in neonatal mice.</a> Laboratory animal science. 49, 328-330 (1999).</li><li>Bevan, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+heart+failure+in+the+SMNDelta7+model+of+spinal+muscular+atrophy+and+correction+by+postnatal+scAAV9-SMN+delivery.">Early heart failure in the SMNDelta7 model of spinal muscular atrophy and correction by postnatal scAAV9-SMN delivery.</a> Human molecular genetics. 19, 3895-3905 (2010).</li><li>Valori, C. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+delivery+of+scAAV9+expressing+SMN+prolongs+survival+in+a+model+of+spinal+muscular+atrophy.">Systemic delivery of scAAV9 expressing SMN prolongs survival in a model of spinal muscular atrophy.</a> Science translational medicine. 2, (2010).</li><li>Foust, K. D., Poirier, A., Pacak, C. A., Mandel, R. J., Flotte, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+intraperitoneal+or+intravenous+injections+of+recombinant+adeno-associated+virus+type+8+transduce+dorsal+root+ganglia+and+lower+motor+neurons.">Neonatal intraperitoneal or intravenous injections of recombinant adeno-associated virus type 8 transduce dorsal root ganglia and lower motor neurons.</a> Hum Gene Ther. 19, 61-70 (2008).</li><li>Guggenbuhl, P., Grosbois, B., Chales, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gaucher+disease.">Gaucher disease.</a> Joint, bone, spine : revue du rhumatisme. 75, 116-124 (2008).</li><li>Daly, T. M., Vogler, C., Levy, B., Haskins, M. E., Sands, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+gene+transfer+leads+to+widespread+correction+of+pathology+in+a+murine+model+of+lysosomal+storage+disease.">Neonatal gene transfer leads to widespread correction of pathology in a murine model of lysosomal storage disease.</a> Proc Natl Acad Sci U S A. 96, 2296-2300 (1999).</li><li>Daly, T. M., Ohlemiller, K. K., Roberts, M. S., Vogler, C. A., Sands, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+of+systemic+clinical+disease+in+MPS+VII+mice+following+AAV-mediated+neonatal+gene+transfer.">Prevention of systemic clinical disease in MPS VII mice following AAV-mediated neonatal gene transfer.</a> Gene Ther. 8, 1291-1298 (2001).</li><li>Wang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Juvenile+neuronal+ceroid+lipofuscinoses.">Juvenile neuronal ceroid lipofuscinoses.</a> Advances in experimental medicine and biology. 724, 138-142 (2012).</li><li>Forsythe, E., Beales, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bardet-Biedl+syndrome.">Bardet-Biedl syndrome.</a> European journal of human genetics. 21, 8-13 (2013).</li><li>Bevan, A. 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Die Autoren haben nichts zu offenbaren.

Intravaskuläre Abgabe von Mitteln an das ZNS oder im ganzen Körper ist schwer in neonatalen Mausmodellen von Krankheiten. Das beschriebene Protokoll ist eine schnelle, relativ nicht-invasive Weise, intravenös verabreichen Lösungen in neugeborenen Mäusen mit minimalen Anforderungen an die Ausrüstung. Wenn die zeitliche Fläche Vene mit dem bloßen Auge gesehen werden kann, können Injektionen größere Genauigkeit unter Verwendung des Mikroskops und faseroptische Lichtquelle, vor allem für einen ungeübten Injekto...

Lieferung von Therapeutika auf das zentrale Nervensystem (ZNS) in Mausmodellen der pädiatrische Erkrankung bleibt eine Herausforderung. Mäuse, die Modell Neugeborenen Krankheitszustände sind unterdimensioniert und entwicklungs unreif, und kann daher schwer direkt in geeignete Strukturen im ZNS zu injizieren. Intravaskuläre Injektion von therapeutischen Mitteln ist eine nicht-invasive, gut verträgliche Methode, um Zellen, Drogen oder viralen Vektoren an den gesamten Körper einschließlich des CNS 1-5 und Retina 3,5-9 liefern. Zurück Publikationen beschreiben zeitliche Gesicht Veneninjektion mit einem Transilluminator 10,11, ohne Dissektionsmikroskop 11,12 oder zwei Individuen erfordern spritzen 10. Die Injektionstechnik in diesem Protokoll beschrieben ist vorteilhaft, weil eine einzelne Person kann Welpen zu injizieren, und die Lichtquelle zu sehen die zeitliche Vene den Welpen nicht zu berühren, wodurch die Notwendigkeit für chirurgische Band oder das Anbringen eines Welpen auf eine feste Oberflächewie einem Transilluminator 11. Lieferung von Adeno-assoziierten viralen Vektor Serotyp 9 (AAV9) in Mäusen erzeugt robuste Expression in Neuronen und Astrozyten im Gehirn und Rückenmark (Abbildung 1). Intravaskuläre Lieferung von viralen Vektoren in die temporalis superficialis V. facialis wurde zuverlässig in verschiedenen Studien in neugeborenen Mäusen verwendet, um die pädiatrische neuromuskuläre Erkrankung Spinale Muskelatrophie (SMA) 2,4,13,14 behandeln und letztlich erhöhte die Lebensdauer der behandelten Mäuse. Intravaskuläre Injektion von neugeborenen Mäusen auch effektiv zielt auf das periphere Nervensystem und peripheren Organen (Abbildung 2). Nach Injektion von AAV hat Transduktion von Spinalganglien, Leber, Herz, Skelettmuskel, Lunge und Plexus myentericus des Darms beobachtet 1,3,6,7,15. Verbreitete Transduktion des ZNS und Peripherie macht dieses Verfahren ideal für Injektionskrankheiten globalen Expression erforderlichein Transgen, wie Morbus Gaucher 16 und andere lysosomale Speicherkrankheiten 17,18, Batten-Krankheit und verwandte neuronale Zeroidlipofuszinosen, 19 und Bardet-Biedl-Syndrom, einer genetischen Multisystemerkrankung mit Beginn der Symptome in der frühen Kindheit 20 auftritt. Intravaskulärer Injektion in neugeborenen Mäusen sollte auch als ein neuartiges Verfahren zur Modellierung systemweite pädiatrische Erkrankungen angesehen werden. Diese Technik hat den großen Tiermodellen 5,21 übersetzt und intravaskuläre Injektion als eine klinisch akzeptable Methode der Bereitstellung von Therapeutika ist bereits vorhanden. Das aktuelle Protokoll beschreibt eine einfache, effiziente Methode der Bereitstellung von Mitteln, um neugeborenen Mäusen durch die temporalis superficialis Gesicht Vene spätestens postnatalen Tag 2. Injektion kann durch eine einzige abgeschlossen werden, praktiziert einzelnen und ist gut von den beiden Jungtieren und die Dämme toleriert. Pups erleben minimal Not und erholen sich schnell. Importantly werden erfolgreiche Injektion in globalen Auslieferung des verabreichten Mittels führen. Dieses Protokoll ist für die Lieferung von viralen Vektoren, pharmazeutische Mittel oder Zellen neugeborener Mäuse geeignet.

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			<td style="width:119px;">Catalog Number</td>
			<td style="width:188px;">Comments/Description</td>
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			<td height="42" style="height:42px;width:216px;">Thinpro Insulin Syringe</td>
			<td style="width:103px;">Terumo</td>
			<td>SS30M3009</td>
			<td style="width:188px;">3/10cc, 3/8&#34; needle, 30G, 1 per mouse</td>
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			<td height="42" style="height:42px;width:216px;">Evans Blue Dye</td>
			<td style="width:103px;">Sigma-Aldrich</td>
			<td style="width:119px;">E2129</td>
			<td style="width:188px;">Dilute to 1% with 1X Phosphate Buffered Saline&#160;</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cotton Tipped Applicators</td>
			<td style="width:103px;">Fisher Scientific&#160;</td>
			<td style="width:119px;">23-400-101</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fiber Optic Light Source&#160;</td>
			<td style="width:103px;">Fisher Scientific&#160;</td>
			<td style="width:119px;">12-562-36</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Dissecting Microscope</td>
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intravenous injections in neonatal mice

Die intravenöse Injektion ist eine klinisch anwendbare Weise Therapeutika liefern. Für erwachsene Nager und größere Tiere, sind intravenöse Injektionen technisch möglich und Routine. Jedoch können einige Mausmodellen frühen Ausbruch der Krankheit mit einem schnellen Fortschreiten, die Verabreichung von potentiellen Therapien schwierig macht. Die zeitliche (oder Gesichts) Vene nur an das Ohr Knospe anterioren bei Mäusen und ist eindeutig für die ersten zwei Tage nach der Geburt sichtbar auf beiden Seiten des Kopfes mit einem Binokular. Während diesem Fenster können die zeitliche Vene mit einem Volumen von bis zu 50 &amp; mgr; l injiziert werden. Die Injektion ist sicher und gut durch sowohl den Welpen und der Muttertiere vertragen. Eine typische Injektionsverfahren wird innerhalb von 1-2 min, wonach der Welpe ist mit dem Heimkäfig zurück abgeschlossen. Mit dem dritten Tag nach der Geburt die Vene ist schwierig zu visualisieren und das Einspritzverfahren wird technisch unzuverlässig. Diese Technik ist für die Lieferung von Adeno-assoziierten Virus (AAV) vect verwendetors, die wiederum fast körper breit, stabile Transgenexpression für das Leben des Tieres in Abhängigkeit von dem viralen Serotyp ausgewählt ist.

Während einer richtigen Injektion sollte die Vene vorübergehend drehen klar oder blanchieren. Wenn Injizieren färben die gesamte pup sollte blau innerhalb von Sekunden zu drehen. Wenn eine fehlerhafte Einspritzung stattgefunden hat, gibt es oft eine konzentrierte subkutaner Bolus in den Kopf oder Nacken und Injektionsmittel kann aus der Injektionsstelle zu lecken. Unsachgemäße Injektionen können auch im Erscheinungsbild des Blutergüsse um den Hals führen. Pups, die subkutane Injektionen erhalten (dh die...

Watch this Scientific Journal Video about Intravenous Injections in Neonatal Mice at JoVE.com

Intravenous Injections in Neonatal Mice

Tiermodelle der pädiatrischen Erkrankung früh einsetzende und aggressiven Krankheitsprogression zu erleben. Klinisch relevante Therapieabgabe, junge Maus-Modellen kann technisch schwierig sein. Dieses Protokoll beschreibt eine nicht-invasive Methode zur intravenösen Injektion neugeborene Mäuse innerhalb der ersten zwei Tage nach der Geburt des Lebens.

Intravenöse Injektionen in neugeborenen Mäusen

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are ...

Research

JoVE Journal

Biology

57.8K Aufrufe.  Ohio State University.  Tiermodelle der pädiatrischen Erkrankung früh einsetzende und aggressiven Krankheitsprogression zu erleben. Klinisch relevante Therapieabgabe, junge Maus-Modellen kann technisch schwierig sein. Dieses Protokoll beschreibt eine nicht-invasive Methode zur intravenösen Injektion neugeborene Mäuse innerhalb der ersten zwei Tage nach der Geburt des Lebens. 

Serie: Intravenous Injections in Neonatal Mice

Heterotopic Heart Transplantation in Mice

The mouse heterotopic heart transplantation has been used widely since it was introduced by Drs. Corry and Russell in 1973. It is particularly valuable for studying rejection and immune response now that newer transgenic and gene knockout mice are available, and a large number of immunologic reagents have been developed. The heart transplant model is less stringent than the skin transplant models, although technically more challenging. We have developed a modified technique and have completed over 1000 successful cases of heterotopic heart transplantation in mice. When making anastomosis of the ascending aorta and abdominal aorta, two stay sutures are placed at the proximal and distal apexes of recipient abdominal aorta with the donor s ascending aorta, then using 11-0 suture for anastomosis on both side of aorta with continuing sutures. The stay sutures make the anastomosis easier and 11-0 is an ideal suture size to avoid bleeding and thrombosis.
When making anastomosis of pulmonary artery and inferior vena cava, two stay sutures are made at the proximal apex and distal apex of the recipient s inferior vena cava with the donor s pulmonary artery. The left wall of the inferior vena cava and donor s pulmonary artery is closed with continuing sutures in the inside of the inferior vena cava after, one knot with the proximal apex stay suture the right wall of the inferior vena cava and the donor s pulmonary artery are closed with continuing sutures outside the inferior vena cave with 10-0 sutures. This method is easier to perform because anastomosis is made just on the one side of the inferior vena cava and 10-0 sutures is the right size to avoid bleeding and thrombosis. In this article, we provide details of the technique to supplement the video.

The mouse heterotopic heart transplantation model has been proven by many investigators to be an important method for studying mechanisms of rejection and immune response. However, the techniques involved are still challenging. By modifying standard techniques we have had success with more than 1000 transplants.

Heterotope Herztransplantation in Mäuse

The mouse heterotopic heart transplantation has been used widely since it was introduced by Drs. Corry and Russell in 1973. It is particularly valuable ...

Forschung

Biologie

Small Bowel Transplantation In Mice

Since 1990, the development of tacrolimus-based immunosuppression and improved surgical techniques, the increased array of potent immunosuppressive medications, infection prophylaxis, and suitable patient selection helped improve actuarial graft and patient survival rates for all types of intestine transplantation. Patients with irreversible intestinal failure and complications of parenteral nutrition should now be routinely considered for small intestine transplantation. However, Survival rates for small intestinal transplantation have been slow to improve compares increasingly favorably with renal, liver, heart and lung. The small bowel transplantation is still unsatisfactory compared with other organs. Further progress may depend on better understanding of immunology and physiology of the graft and can be greatly facilitated by animal models. A wider use of mouse small bowel transplantation model is needed in the study of immunology and physiology of the transplantation gut as well as efficient methods in diagnosing early rejection. However, this model is limited to use because the techniques involved is an extremely technically challenging. We have developed a modified technique. When making anastomosis of portal vein and inferior vena cava, two stay sutures are made at the proximal apex and distal apex of the recipient s inferior vena cava with the donor s portal vein. The left wall of the inferior vena cava and donor s portal vein is closed with continuing sutures in the inside of the inferior vena cava after, after one knot with the proximal apex stay suture the right wall of the inferior vena cava and the donor s portal vein are closed with continuing sutures outside the inferior vena cave with 10-0 sutures. This method is easier to perform because anastomosis is made just on the one side of the inferior vena cava and 10-0 sutures is the right size to avoid bleeding and thrombosis.  In this article, we provide details of the technique to supplement the video.

The mouse small bowel transplantation model has been recognized as an important tool to study mechanismes of immune rejection and screen new immunosuppressive drugs. However, this model is limited to use because the techniques involved is an extremely technically challenge. Now we introduce the modified technique.

Dünndarmtransplantation In Mice

Since 1990, the development of tacrolimus-based immunosuppression and improved surgical techniques, the increased array of potent immunosuppressive ...

Transverse Aortic Constriction in Mice

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model for pressure overload-induced cardiac hypertrophy and heart failure.1 TAC initially leads to compensated hypertrophy of the heart, which often is associated with a temporary enhancement of cardiac contractility. Over time, however, the response to the chronic hemodynamic overload becomes maladaptive, resulting in cardiac dilatation and heart failure.2 The murine TAC model was first validated by Rockman et al.1, and has since been extensively used as a valuable tool to mimic human cardiovascular diseases and elucidate fundamental signaling processes involved in the cardiac hypertrophic response and heart failure development. When compared to other experimental models of heart failure, such as complete occlusion of the left anterior descending (LAD) coronary artery, TAC provides a more reproducible model of cardiac hypertrophy and a more gradual time course in the development of heart failure. Here, we describe a step-by-step procedure to perform surgical TAC in mice. To determine the level of pressure overload produced by the aortic ligation, a high frequency Doppler probe is used to measure the ratio between blood flow velocities in the right and left carotid arteries.3, 4 With surgical survival rates of 80-90%, transverse aortic banding is an effective technique of inducing left ventricular hypertrophy and heart failure in mice.

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model to study mechanisms underlying cardiac hypertrophy and heart failure development. Here, we describe procedures to constrict the aorta to create a reproducible degree of cardiac hypertrophy in mice.

Quer Aorten-Verengung in Mäuse

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model for pressure overload-induced cardiac hypertrophy and heart ...

Ambulatory ECG Recording in Mice

Telemetric ECG recording in mice is essential to understanding the mechanisms behind arrhythmias, conduction disorders, and sudden cardiac death. Although the surface ECG is utilized for short-term measurements of waveform intervals, it is not practical for long-term studies of heart rate variability or the capture of rare episodes of arrhythmias. Implantable ECG telemeters offer the advantages of simple surgical implantation, long-term recording of electrograms in ambulatory mice, and scalability with simultaneous recordings of multiple animals. Here, we present a step-by-step guide to the implantation of telemeters for ambulatory ECG recording in mice. Careful adherence to aseptic technique is required for favorable survival results with the possibility of implantation and recording from weeks to months. Thus, implantable ECG telemetry is a valuable tool for detection of critical information on cardiac electrophysiology in ambulatory animal models such as the mouse. 

Telemetric ECG has emerged as an essential tool in evaluating animal models for cardiac arrhythmias and sudden cardiac death. Here, we present a stepwise guide to telemetric ECG recordings for application in long-term ambulatory ECG monitoring in mice.

Ambulante EKG-Aufzeichnung in Mäuse

Telemetric ECG recording in mice is essential to understanding the mechanisms behind arrhythmias, conduction disorders, and sudden cardiac death. Although ...

Intravenous Microinjections of Zebrafish Larvae to Study Acute Kidney Injury

In this video article we describe a zebrafish model of AKI using gentamicin as the nephrotoxicant.  The technique consists of intravenous microinjections on 2 dpf zebrafish. This technique represents an efficient and rapid method to deliver soluble substances into the bloodstream of zebrafish larvae, allowing for the injection of 15-20 fish per hour. In addition to AKI studies, this microinjection technique can also be used for other types of experimental studies such as angiography.  We provide a detailed protocol of the technique from equipment required to visual measures of decreased kidney function. In addition, we also demonstrate the process of fixation, whole mount immunohistochemistry with a kidney tubule marker, plastic embedding and sectioning of the larval zebrafish. We demonstrate that zebrafish larvae injected with gentamicin show morphological features consistent with AKI: edema, loss of cell polarity in proximal tubular epithelial cells, and morphological disruption of the tubule.

We describe a technique of microinjecting the aminoglycoside, gentamicin, into 2 days post-fetilization (dpf) zebrafish larvae to induce acute kidney injury (AKI). We also describe a method for whole mount immunohistochemistry, plastic embedding and sectioning of zebrafish larvae to visualize the AKI mediated damage.

Intravenöse Mikroinjektionen mit Zebrabärblingembryonen zu Acute Kidney Injury Study

In this video article we describe a zebrafish model of AKI using gentamicin as the nephrotoxicant.  The technique consists of intravenous microinjections ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

Isolierung und Kultivierung von pulmonaler Endothelzellen aus neugeborenen Mäusen

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes.	
The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.

Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays.

Isolation und Kultur der Neugeborenen-Maus-Kardiomyozyten

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

Isolation of Pulmonary Artery Smooth Muscle Cells from Neonatal Mice

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling pathways involved in vascular smooth muscle contraction in PASMC from fetal sheep. While sheep make an excellent model of term pulmonary hypertension, they are very expensive and lack the advantage of genetic manipulation found in mice. Conversely, the inability to isolate PASMC from mice was a significant limitation of that system. Here we described the isolation of primary cultures of mouse PASMC from P7, P14, and P21 mice using a variation of the previously described technique of Marshall et al.26 that was previously used to isolate rat PASMC. These murine PASMC represent a novel tool for the study of signaling pathways in the neonatal period. Briefly, a slurry of 0.5% (w/v) agarose + 0.5% iron particles in M199 media is infused into the pulmonary vascular bed via the right ventricle (RV). The iron particles are 0.2 &#956;M in diameter and cannot pass through the pulmonary capillary bed. Thus, the iron lodges in the small pulmonary arteries (PA). The lungs are inflated with agarose, removed and dissociated. The iron-containing vessels are pulled down with a magnet. After collagenase (80 U/ml) treatment and further dissociation, the vessels are put into a tissue culture dish in M199 media containing 20% fetal bovine serum (FBS), and antibiotics (M199 complete media) to allow cell migration onto the culture dish. This initial plate of cells is a 50-50 mixture of fibroblasts and PASMC. Thus, the pull down procedure is repeated multiple times to achieve a more pure PASMC population and remove any residual iron. Smooth muscle cell identity is confirmed by immunostaining for smooth muscle myosin and desmin.

We have developed a novel and reproducible technique to isolate primary cultures of pulmonary artery smooth muscle cells (PASMC) from mice as young as P7, thereby allowing better study of the signaling pathways involved in neonatal smooth muscle cell contraction and relaxation.

Isolation der Lungenarterie glatten Muskelzellen aus neugeborenen Mäusen

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling ...

In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice

This report describes a step-by-step guide to the technique of acute intrathecal needle injections in a noninvasive manner, i.e. independent of catheter implantation. The technical limitation of this surgical technique lies in the finesse of the hands. The injection is rapid, especially for a trained experimenter, and since tissue disruption with this technique is minimal, repeated injections are possible; moreover immune reaction to foreign tools (e.g. catheter) does not occur, thereby giving a better and more specific read out of spinal cord modulation. Since the application of the substance is largely limited to the target region of the spinal cord, drugs do not need to be applied in large dosages, and more importantly unwanted effects on other tissue, as observed with a systemic delivery, could be circumvented1,2. Moreover, we combine this technique with in vivo transfection of nucleic acid with the help of polyethylenimine (PEI) reagent3, which provides tremendous versatility for studying spinal functions via delivery of pharmacological agents as well as gene, RNA, and protein modulators.

In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice

This report describes a simple and rapid technique of intrathecal needle puncture for a localized transfection of siRNA in the lumbar spinal cord in mouse under short lasting light anesthesia.

In vivo siRNA-Transfektion und Gene Knockdown im Rückenmark über Rapid-Nichtinvasive Lendenwirbel intrathekale Injektionen in Mäuse

This report describes a step-by-step guide to the technique of acute intrathecal needle injections in a noninvasive manner, i.e. independent of catheter ...

Isolation of Neonatal Extrahepatic Cholangiocytes

The intra and extrahepatic bile ducts of the liver are developmentally distinct, and may be differentially affected by certain diseases. However, differences between intra and extrahepatic cholangiocytes, and between neonatal and adult cells, are not well understood.
Methods for the isolation of cholangiocytes from intrahepatic bile ducts are well established1-4. Isolation of extrahepatic ductal cells, especially from the neonate, has not yet been described, although this would be of great benefit in understanding the differences between distinct cholangiocyte populations and in studying diseases such as biliary atresia that appear to target the extrahepatic ducts. Described here is an optimized technique to isolate both neonatal and adult mouse extrahepatic bile duct cells. This technique yields a pure cell population with minimal contamination from mesenchymal cells like fibroblasts.
This method is based on the removal of the extrahepatic ducts and gallbladder, followed by meticulous dissection and scraping to remove fat and fibroblast layers. Structures are embedded in thick layers of collagen and cultured for approximately 3 weeks to allow outgrowth of cholangiocytes in monolayers, which can then be trypsinized and re plated for experimental use.

A technique to isolate cholangiocytes from the extrahepatic bile ducts of neonatal mice is described. The ducts are meticulously dissected, and then cells are isolated by outgrowth in thick collagen gels. This method provides a useful tool for studying extrahepatic bile duct development and pathology.