The authors would like to acknowledge the support of NIH/NIDDK K12 DK100014 (Lan Lu), the Case Comprehensive Cancer Center (NIH/NCI P30 CA43703), and the Clinical and Translation Science Collaborative of Cleveland (NIH/NCATS UL1 TR000439).

Alle Tierstudien wurden nach den genehmigten Protokollen des örtlichen Tierpflege- und -nutzungsausschusses (IACUC) an der Case Western Reserve University durchgeführt. Die Dauer des nachstehend beschriebenen chirurgischen Verfahrens beträgt ca. 45-60 min. Die MRT-Prozedur selbst beträgt ca. 15 min für jeden Zeitpunkt. 1. Anästhesie <ol><li> Anästhesieren Sie die Ratte in der Isoflurankammer, die auf 2% Isofluran eingestellt ist, um mit dem Tier gemischt zu werden, um die Tierbehandlung und die Zurückhaltung zu erleichtern, bevor die injizierbare Anästhesie intraperitoneal verabreicht wird. </li><li> Nach 3-5 min Exposition gegenüber Isofluran ist zu prüfen, ob das Tier anästhesiert ist und keine Reaktion auf Zehenknecht zeigt. </li><li> Weitere sedierte Ratte mit einer intraperitonealen Injektion einer Mischung aus Xylazin und Ketamin: 75 mg / kg Ketamin / 10 mg / kg Xylazin. Bei der Durchführung von intra-peritonealen Injektionen, ziehen Sie die Nadel zurück, um sicherzustellen, dass Teile des Darms oder anderer lebenswichtiger Organe nicht punktiert wurden. </li><li> ich2 mg / kg Bupivacain subkutan an der Stelle des Einschnitts injizieren, um eine topische Schmerzlinderung zu liefern. </li></ol> 2. Vorbereitung des chirurgischen Bereichs <ol><li> Sterilisieren Sie chirurgische Instrumente und Vorräte, bevor sie für die Chirurgie verwendet werden und legen Sie auf dem chirurgischen Pad für Sterilität. Die meisten Instrumente und Zubehör können autoklaviert und wiederverwendet werden. </li><li> Verwenden Sie sterile Handschuhe für alle chirurgischen Eingriffe. </li><li> Verwenden Sie ein elektrisches Rasiermesser, um das Fell von der rechten Seite des Tieres zu rasieren. Rasiere das Tier von der Unterseite des Brustkorbes bis zur Oberseite des Hinterbeins, was einen großen haarfreien Bereich für den Schnitt bietet. </li><li> Lege das Tier auf eine sterile chirurgische Auflage, um den desinfizierten Bereich von den umliegenden Gebieten zu isolieren. </li><li> Die Haut mit einem Desinfektionsmittel wie Povidonjod oder Betadin scheuern. Starten Sie das Schrubben in der Mitte des chirurgischen Geländes und bewegen Sie sich kreisförmig nach außen. Wiederholen Sie mindestens dreimal mit einem neuen Tuch von Povidon Jod oder Betadin. </Li&gt; <li> Schrubben Sie die chirurgische Stelle mit 70% Alkoholtupfern, bis die Haut klar ist, da Jod giftig ist, wenn es absorbiert wird. </li></ol> 3. Chirurgische Vorgehensweise <ol><li> Halten Sie diese Vorgehensweise unter aseptischen Bedingungen ein. </li><li> Das anästhesierte Tier auf ein warmes Heizbett in der linken seitlichen Dekubitusposition bringen, wobei die rechte Flanke nach oben zeigt. HINWEIS: Es ist darauf zu achten, dass die Kerntemperatur des Tieres bei 35-37 ° C gehalten wird, um eine Unterkühlung zu verhindern. Dieses Erwärmungsbett sollte auch nach Bedarf sterilisiert werden, um aseptische Bedingungen aufrechtzuerhalten. </li><li> Fühlen Sie sich für den Brustkorb und machen Sie einen kleinen 2-3 cm rechten dorsalen retroperitonealen Einschnitt mit einer sterilen Größe 10 Skalpell Klinge beginnend am unteren Rand des Brustkorbes. </li><li> Lege sterile Gaze in Längsrichtung auf beiden Seiten des Schnittes. </li><li> Das subkutane Gewebe, Fett und Muskeln zerlegen, um die Bauchhöhle zu visualisieren und zugänglich zu machen. Verwenden Sie eine gekrümmte Mayo-Schere, um tieferes Penet zuzulassenRation in Wunde und schneiden dicke Gewebe. </li><li> Sobald die Leber deutlich sichtbar und zugänglich ist, verwenden Sie stumpfe Pinzette, um die Leber nach oben zurückzuziehen. </li><li> Mit einem anderen Paar von stumpfen Pinzetten in der anderen Hand, stellen Sie die richtige Niere, so dass es sitzt gerade außerhalb der Bauchhöhle. </li><li> Benutze den Zeiger Finger und Daumen der linken Hand, um die Niere in Position zu halten. Mit der rechten Hand langsam und stetig 0,1 ml UTI89 E. coli- Lösung (Konzentration zwischen 1 x 10 8 -1 x 10 9 ) aus einer sterilen Spritze in das Nierenbecken (das als weiße Blase erscheint) einspritzen 15 . HINWEIS: Den Bakterientiter wie in Referenz 15 beschrieben vorbereiten. </li><li> Legen Sie einen Streifen absorbierbaren Hämostat über die Nadel, um den Ausfluss des Inokulums in das Peritoneum zu verhindern. Ziehen Sie langsam die Nadel aus dem Nierenbecken. HINWEIS: Es ist darauf zu achten, dass die umlaufenden Gewebe nicht durchbohrt oder infiziert werden, was zu einem Ziel-Infecti führtOns und / oder komplikationen. </li><li> Verwenden Sie eine größere Spritze, um die Niere gründlich mit normaler Kochsalzlösung zu spülen, bevor Sie sie wieder in die Bauchhöhle legen. </li></ol> 4. Nähte Anmerkung: Naht, die in Geweben begraben wird, sollte 4-0 nicht resorbierbare geflochtene Nähte sein. Für Körperoberflächen können absorbierbare oder monofile Nähte verwendet werden. <ol><li> Setzen Sie die Nähte gleichmäßig und so nah wie möglich an die Gewebekante, um eine Verstopfung des Blutflusses zu verhindern. Typischerweise ist nicht mehr als 0,3 cm von der Kante notwendig. </li><li> Fassen Sie die Haut an und verjüngen Sie sie leicht mit einem Paar feiner Zahnpinzette und drehen Sie den Nadelhalter in eine pronierte Position in Vorbereitung für das Durchstechen der Haut. </li><li> Führen Sie die Nadel durch die volle Dicke der Haut, indem Sie das Handgelenk anspannen, um die Nadel zu drehen und es durch die Haut zu führen. </li><li> Wiederholen Sie diesen Vorgang für die Hautkante, die dem Einzelnen am nächsten liegt. </li><li> SpannenDie Nähte genug, um den Gewebekanten entgegenzutreten. Je enger wird die Blutversorgung versperren, langsame Wundheilung und kann zu Dehiszenzen führen. </li><li> Binden Sie die Naht mit quadratischen Knoten, als ob es eine einfache unterbrochene Stich wäre, außer dass nur der kurze Strang geschnitten wird, so dass ein 3-4 mm Schwanz. </li><li> Nachdem die erste Masche abgebunden ist, bereite ich mich vor, um eine zweite Masche ca. 3 mm von der ersten weg zu setzen und die laufende Naht fortzusetzen. </li><li> Sobald das Ende des Einschnitts erreicht ist, ziehen Sie nicht die letzte Stich vollständig durch. Verwenden Sie stattdessen die Schlaufe, die mit dem Nadelhalter hier als Kurzstrang gehalten wird, um das distale Ende des Nahtverschlusses zu verkleben. </li><li> Mit Instrumentenbindungen, binden Sie die Naht mit quadratischen Knoten. Dies führt dazu, dass 3 Stränge aus dem fertigen Knoten am distalen Ende aufstehen. </li></ol> 5. Tierrückgewinnung <ol><li> Injizieren Sie 2,1 mg / kg Yohimbin intra-peritoneal nach der Operation, um die Anästhesie umzukehren. EINNimals wird erwartet, um vollständig von der chirurgischen Prozedur nach 3-5 Stunden zu erholen. </li><li> Halten Sie das Tier auf einem Heizkissen (um Hypothermie zu vermeiden) und saugfähige weiche Bettwäsche nach der Operation. </li><li> Rehydrierung mit oralen oder parenteralen Flüssigkeiten bereitstellen, bis es zur normalen Fütterung zurückkehren kann (innerhalb von 24 Stunden). Injektion von etwa 0,6 ml normaler Kochsalzlösung sofort nach der Operation, um die Dehydratationseffekte zu begrenzen. </li><li> Bereitstellung von Analgesie, wie im IACUC zugelassenen Tierpflege- und Verwendungsprotokoll beschrieben. 5 mg / kg Carprofen subkutan zur Schmerztherapie injizieren. </li><li> Überwachen Sie die Schnitte regelmäßig auf Schwellungen, Exsudate, Schmerzen oder Dehiszenzen. </li></ol> 6. Validierung mittels Magnetresonanztomographie <ol><li> Führen Sie i n vivo MRT-Experimente auf Hochfeld-Kleintier-MRT-Scannern durch. </li><li> Anästhesie mit 3% Isofluran in Sauerstoff induzieren und das Tier mit der rechten Niere bei Isocenter im MRT-Scanner positionieren und entsprechendeAte Hochfrequenzspule. Bereitstellung von Tieren mit 1-2% Isofluran-Anästhesie kontinuierlich während des Imaging-Verfahrens über einen Nosecone. HINWEIS: Für die in Fig. 1 gezeigten in vivo- MRI-Bilder wurde eine Rattenvolumenspule (Innendurchmesser = 72 mm) verwendet. </li><li> Verwenden Sie ein Tierüberwachungs- und Kontrollsystem, um die Atmungsrate jedes Tieres (40-60 Atemzüge / min) und die Kernkörpertemperatur (35 ± 1 ° C) aufrechtzuerhalten. </li><li> Verwenden Sie eine Multi-Slice-, Multi-Echo-Spin-Echo-MRT-Erfassung, um hochauflösende, axiale T2-gewichtete Bilder sowohl der infizierten als auch der Kontroll-Nieren zu erhalten. Typische MRT-Erfassungsparameter sind Wiederholungszeit = 5.000 ms, Echozeit = 40 ms, Scheibendicke = 2,0 mm, flache Raumauflösung = 200 μm, 3 Signaldurchschnitte und eine Erfassungszeit von 8 min. </li></ol>

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The authors have no other disclosures.

Aufsteigende akute Pyelonephritis bei Nagetieren (dh Mäusen und Ratten) kann durch transurethrale Katheterisierung hergestellt werden. 16 , 17 , 18 Diese transurethrale Infektionsmethode ist insofern vorteilhaft, als sie nicht invasiv ist und die menschliche Pathophysiologie der aufsteigenden Infektion nachahmt. 17 , 18 , 19 ...

Nagetier-Modelle wurden verwendet, um zahlreiche menschliche Erkrankungen Manifestationen, einschließlich Pyelonephritis und Harnwegsinfektionen (UTI) zu studieren. UTIs sind ein globales Gesundheitsproblem und können Kinder, Männer und Frauen aller Altersstufen beeinflussen. 1 , 2 , 3 Die anfängliche Manifestation von UTIs umfasst Zystitis, und wenn die Infektion entlang des Harnleiters steigt, kann eine Niereninfektion (Pyelonephritis) folgen. Gleichzeitig nähert sich die Prävalenz von Diabetes 400 Millionen Menschen weltweit. 4 , 5 Wichtig ist, dass die UTI-Inzidenz bei Patienten, die fettleibig sind oder einen Typ-2-Diabetes mellitus haben, bis zu 4-mal höher sein kann, was zu einem erhöhten Risiko einer rezidivierenden UTI-Infektion (RUTI), Sepsis, Nierenfibrose aus Pyelonephritis und Blasenfunktionsstörungen führt. 6 , 7 , 8 NagetierModelle sind wichtig für das Studium von UTIs, da aktuelle Antibiotika-Therapien eine nachhaltige, präventive Reaktion nur in einer Teilmenge von UTI-Patienten produzieren. Zur Verbesserung der klinischen UTI-Pflege sind die wichtigsten Schritte, um den Mechanismus von RUTI und seine pathophysiologischen Prozesse von akuten Infektionen bis hin zur Entzündung der Fibrose sowie die Auswirkungen von Typ-2-Diabetes mellitus zu verstehen. Das Ziel der Verbesserung der Tiermodelle ist die Entwicklung von Techniken, die eine genauere Bewertung der Krankheitsprogression und therapeutische Interventionen ermöglichen. Es wurden verschiedene Ansätze verwendet, um Pyelonephritis bei Ratten und / oder Mäusen zu induzieren, um die Pathophysiologie der Nierenschäden, die Wirkung der Antibiotika-Behandlung und andere Aspekte des natürlichen Verlaufs von UTIs zu untersuchen. Eine gemeinsame Herangehensweise, um retrograde UTI zu etablieren, ist die transurethrale Katheterisierung. 10 , 11 , 12 , 13 &lt;/ Sup&gt; Diese Methode führt Bakterien über die Harnröhre in die Harnblase der betäubten Tiere ein. Während diese Technik die menschliche Pyelonephritis genau simuliert, kann die tatsächliche Inzidenz und Größe der Pyelonephritis-Infektion sehr variabel sein, da mehrere Faktoren, einschließlich des Mangels an spontanem ureterischem Reflux oder Harnverhalten während oder unmittelbar nach der Inokulation. 11 Als Ergebnis kann die experimentelle Variabilität bei der Induktion einer aufsteigenden Pyelonephritis-Infektion den Nutzen dieses Modells einschränken, um Niereninfektionen sowie therapeutische Strategien zu untersuchen. Dieser Bericht beschreibt ein chirurgisches Pyelonephritis-Rattenmodell, bei dem E. coli direkt in die Rattenniere injiziert wird. Trotz dieses Rattenmodells, das invasiv ist, kann die Menge an E. coli, die an die Niere abgegeben wird, effektiv kontrolliert werden, was eine robuste Niereninfektion und eine Entzündung ermöglicht. 14 In diesem Verfahren beschreiben wir auchWie diese induzierten Niereninfektionen in Längsrichtung mit in vivo Magnetresonanztomographie (MRT) überwacht werden können.

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			<td height="21" style="height:21px;width:216px;">Absorbing Pad</td>
			<td style="width:195px;">Fisher</td>
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			<td height="21" style="height:21px;">Latex Surgical Gloves</td>
			<td>Henry Schein Animal Health</td>
			<td>21540</td>
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			<td height="21" style="height:21px;width:216px;">Curved Mayo Scissors</td>
			<td style="width:195px;">Fisher</td>
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			<td height="21" style="height:21px;width:216px;">Straight Blunt Foreceps</td>
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			<td height="21" style="height:21px;width:216px;">Sterile Scalpel Blades</td>
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			<td height="21" style="height:21px;width:216px;">1mL Luer-Lok Syringe</td>
			<td style="width:195px;">BD Biosciences</td>
			<td>309628</td>
			<td>For bacterial injections</td>
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		<tr height="21">
			<td height="21" style="height:21px;">20mL Luer-Lok Syringe&#160;</td>
			<td style="width:195px;">BD Biosciences</td>
			<td>301031</td>
			<td>For saline wash</td>
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			<td height="21" style="height:21px;width:216px;">Hemostat</td>
			<td style="width:195px;">Seneca Medical</td>
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			<td height="21" style="height:21px;">23 G 3/4 in. Needle&#160;</td>
			<td style="width:195px;">BD Biosciences</td>
			<td style="width:168px;">305143</td>
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			<td height="21" style="height:21px;">30 G 1 in. Needle&#160;</td>
			<td style="width:195px;">BD Biosciences</td>
			<td style="width:168px;">305128</td>
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			<td height="21" style="height:21px;">U-100 Insulin Syringe</td>
			<td>Exel International&#160;</td>
			<td style="width:168px;">25846</td>
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			<td height="21" style="height:21px;">Isoflurane</td>
			<td>Henry Schein Animal Health</td>
			<td>050033</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xylazine</td>
			<td>Henry Schein Animal Health</td>
			<td>33197</td>
			<td>Inject IP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ketamine</td>
			<td>Patterson Vetrinary&#160;</td>
			<td>07-881-9413</td>
			<td>Inject IP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Yohimbine (Atipamezole)</td>
			<td style="width:195px;">Patterson Vetrinary&#160;</td>
			<td>07-867-7097</td>
			<td>Inject IP after surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bupivacaine (Marcaine)</td>
			<td style="width:195px;">Patterson Vetrinary&#160;</td>
			<td>07-890-4584</td>
			<td>Inject SQ at site of incision&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Carprofen (Rimadyl)</td>
			<td style="width:195px;">Patterson Vetrinary&#160;</td>
			<td>07-844-7425</td>
			<td>Should be kept at 4 &#7506;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">4-0 Chromic Gut Suture</td>
			<td style="width:195px;">Ethicon Inc.</td>
			<td>U203H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-0 Braided Vicryl Suture</td>
			<td>Ethicon Inc.</td>
			<td>J304H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1mL SubQ Syringe</td>
			<td>BD Biosciences</td>
			<td>309597</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">E. coli&#160; UTI89 or CFT073</td>
			<td style="width:195px;">ATCC</td>
			<td style="width:168px;">700928</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Surgicel Absorbable Hemostat</td>
			<td style="width:195px;">Ethicon Inc.</td>
			<td>ETH1951CS&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biospec 9.4T MRI&#160;</td>
			<td style="width:195px;">Bruker&#160;</td>
			<td style="width:168px;">94/20 USR</td>
			<td></td>
		</tr>
	</tbody>
</table>

intrarenal injection of escherichia coli in a rat model of pyelonephritis

Pyelonephritis is a bacterial infection of the kidney and is most commonly caused by Escherichia coli. Recurrent infections can cause significant renal inflammation and fibrosis ultimately resulting in declining kidney function. Before improved clinical management and prevention of pyelonephritis can be instituted, a reliable animal model must be established in order to study the mechanisms of progression, recurrence, and therapeutic efficacy. The transurethral infection model closely mimics human pyelonephritis but exhibits considerable variation due to its reliance on urethral reflux to transport the bacteria to the kidney. Herein, a detailed surgical protocol for performing bacterial injections into the rat renal pelvis is provided and confirmed by non-invasive Magnetic Resonance Imaging (MRI). Using this protocol, animals receive direct exposure to a desired concentration of E. coli bacteria and can fully recover from the surgical procedure with adequate post-operative care. This facilitates subsequent longitudinal MRI assessments of the experimental animal models for comparison with saline (sham) controls. Using this direct delivery approach, the severity of infection is controllable and applicable for mechanistic studies of progression as well as development of novel treatment strategies.

Medizinische Bildgebungsverfahren bieten die Möglichkeit, die UTI und die therapeutische Wirksamkeit nicht invasiv zu beurteilen. Daher wurde MRT verwendet, um die Induktion einer akuten Infektion nach der Injektion von 1-2 x 10 7 UTI89 E. coli zu validieren und die Veränderungen in der Niere vor und nach der Operation zu visualisieren. Abbildung 1a-b zeigt eine zunehmend zunehmende Region der Niereninfektion (gelbe Pfeile). MRT-Bilder, die für jed...

Watch this Scientific Journal Video about Intrarenal Injection of Escherichia coli in a Rat Model of Pyelonephritis at JoVE.com

Intrarenal Injection of Escherichia coli in a Rat Model of Pyelonephritis

This manuscript describes a rat surgical model of pyelonephritis using direct intra-renal infection by Escherichia coli into the renal pelvis. The experimental procedure can be utilized to study the pathogenesis of pyelonephritis as well as the associated inflammation and fibrosis.

Intrarenale Injektion von<em&gt; Escherichia coli</em&gt; In einem Rattenmodell der Pyelonephritis

Pyelonephritis is a bacterial infection of the kidney and is most commonly caused by Escherichia coli. Recurrent infections can cause significant renal ...

intrarenal-injection-escherichia-coli-rat-model

Research

JoVE Journal

Medicine

Intrarenal Injection of Escherichia coli in a Rat Model of Pyelonephritis

7.9K Aufrufe.  Case Western Reserve University. This manuscript describes a rat surgical model of pyelonephritis using direct intra-renal infection by Escherichia coli into the renal pelvis. The experimental procedure can be utilized to study the pathogenesis of pyelonephritis as well as the associated inflammation and fibrosis.

Serie: Intrarenal Injection of Escherichia coli in a Rat Model of Pyelonephritis

Coronary Artery Ligation and Intramyocardial Injection in a Murine Model of Infarction

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo. With the advent of genetic modifications to the whole organism or the myocardium and an array of biological and/or synthetic materials, there is great potential for any combination of these to assuage the extent of acute ischemic injury and impede the onset of heart failure pursuant to myocardial remodeling. 
Here we present the methods and materials used to reliably perform this microsurgery and the modifications involved for temporary (with reperfusion) or permanent coronary artery occlusion studies as well as intramyocardial injections. The effects on the heart that can be seen during the procedure and at the termination of the experiment in addition to histological evaluation will verify efficacy. 
Briefly, surgical preparation involves anesthetizing the mice, removing the fur on the chest, and then disinfecting the surgical area. Intratracheal intubation is achieved by transesophageal illumination using a fiber optic light. The tubing is then connected to a ventilator. An incision made on the chest exposes the pectoral muscles which will be cut to view the ribs. For ischemia/reperfusion studies, a 1 cm piece of PE tubing placed over the heart is used to tie the ligature to so that occlusion/reperfusion can be customized. For intramyocardial injections, a Hamilton syringe with sterile 30gauge beveled needle is used. When the myocardial manipulations are complete, the rib cage, the pectoral muscles, and the skin are closed sequentially. Line block analgesia is effected by 0.25% marcaine in sterile saline which is applied to muscle layer prior to closure of the skin. The mice are given a subcutaneous injection of saline and placed in a warming chamber until they are sternally recumbent. They are then returned to the vivarium and housed under standard conditions until the time of tissue collection. At the time of sacrifice, the mice are anesthetized, the heart is arrested in diastole with KCl or BDM, rinsed with saline, and immersed in fixative. Subsequently, routine procedures for processing, embedding, sectioning, and histological staining are performed. 
Nonsurgical intubation of a mouse and the microsurgical manipulations described make this a technically challenging model to learn and achieve reproducibility. These procedures, combined with the difficulty in performing consistent manipulations of the ligature for timed occlusion(s) and reperfusion or intramyocardial injections, can also affect the survival rate so optimization and consistency are critical.

Numerous genetic manipulations and/or intramyocardial injections of genes, proteins, cells, and/or biomaterials are superimposed upon the dimension of time in studies of acute ischemia/ reperfusion injury and chronic remodeling in mice. This video illustrates the microsurgical procedures for ischemia/reperfusion, permanent coronary artery ligation, and intramyocardial injection studies.

Koronare Ligation und intramyokardialen Injection in einem Mausmodell der Infarkt

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo.  With the advent of genetic modifications to ...

Forschung

Medizin

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

Multimodale Bildgebung von Angiogenese in einem Nude Rattenmodell der Brustkrebs Knochenmetastasen mittels Magnetresonanztomographie, volumetrische Computertomographie und Ultraschall

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

Rat Model of Blood-brain Barrier Disruption to Allow Targeted Neurovascular Therapeutics

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain barrier1. The barrier selectively excludes molecules from crossing between the blood and the brain based upon their size and charge. This function can impede the delivery of therapeutics for neurological disorders. A number of chemotherapeutic drugs, for example, will not effectively cross the blood-brain barrier to reach tumor cells2. Thus, improving the delivery of drugs across the blood-brain barrier is an area of interest.
The most prevalent methods for enhancing the delivery of drugs to the brain are direct cerebral infusion and blood-brain barrier disruption3. Direct intracerebral infusion guarantees that therapies reach the brain; however, this method has a limited ability to disperse the drug4. Blood-brain barrier disruption (BBBD) allows drugs to flow directly from the circulatory system into the brain and thus more effectively reach dispersed tumor cells. Three methods of barrier disruption include osmotic barrier disruption, pharmacological barrier disruption, and focused ultrasound with microbubbles. Osmotic disruption, pioneered by Neuwelt, uses a hypertonic solution of 25% mannitol that dehydrates the cells of the blood-brain barrier causing them to shrink and disrupt their tight junctions. Barrier disruption can also be accomplished pharmacologically with vasoactive compounds such as histamine5 and bradykinin6. This method, however, is selective primarily for the brain-tumor barrier7. Additionally, RMP-7, an analog of the peptide bradykinin, was found to be inferior when compared head-to-head with osmotic BBBD with 25% mannitol8. Another method, focused ultrasound (FUS) in conjunction with microbubble ultrasound contrast agents, has also been shown to reversibly open the blood-brain barrier9. In comparison to FUS, though, 25% mannitol has a longer history of safety in human patients that makes it a proven tool for translational research10-12.
In order to accomplish BBBD, mannitol must be delivered at a high rate directly into the brain's arterial circulation. In humans, an endovascular catheter is guided to the brain where rapid, direct flow can be accomplished. This protocol models human BBBD as closely as possible. Following a cut-down to the bifurcation of the common carotid artery, a catheter is inserted retrograde into the ECA and used to deliver mannitol directly into the internal carotid artery (ICA) circulation. Propofol and N2O anesthesia are used for their ability to maximize the effectiveness of barrier disruption13. If executed properly, this procedure has the ability to safely, effectively, and reversibly open the blood-brain barrier and improve the delivery of drugs that do not ordinarily reach the brain 8,13,14.

Blood-brain barrier disruption aids the delivery of certain drugs to the brain. Mannitol delivered intra-arterially shrinks cells surrounding blood vessels in order to physically disrupt the barrier.

Rat Modell der Blut-Hirn-Schranke Störung zu Neurovascular Therapeutics Gezielte zulassen

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain ...

A Model of Chronic Nutrient Infusion in the Rat

Chronic exposure to excessive levels of nutrients is postulated to affect the function of several organs and tissues and to contribute to the development of the many complications associated with obesity and the metabolic syndrome, including type 2 diabetes. To study the mechanisms by which excessive levels of glucose and fatty acids affect the pancreatic beta-cell and the secretion of insulin, we have established a chronic nutrient infusion model in the rat. The procedure consists of catheterizing the right jugular vein and left carotid artery under general anesthesia; allowing a 7-day recuperation period; connecting the catheters to the pumps using a swivel and counterweight system that enables the animal to move freely in the cage; and infusing glucose and/or Intralipid (a soybean oil emulsion which generates a mixture of approximately 80% unsaturated/20% saturated fatty acids when infused with heparin) for 72 hr. This model offers several advantages, including the possibility to finely modulate the target levels of circulating glucose and fatty acids; the option to co-infuse pharmacological compounds; and the relatively short time frame as opposed to dietary models. It can be used to examine the mechanisms of nutrient-induced dysfunction in a variety of organs and to test the effectiveness of drugs in this context.

A protocol for chronic infusions of glucose and Intralipid in rats is described. This model can be used to study the impact of nutrient excess on organ function and physiological parameters.

Ein Modell der chronischen Nährstoff Infusion in der Ratte

Chronic exposure to excessive levels of nutrients is postulated to  affect the function of several organs and tissues and to contribute to the  ...

Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on animal models. The identification of PD-associated genes has led to the development of genetic PD models. Most transgenic &#945;-SYN mouse models develop gradual &#945;-SYN pathology but fail to display clear dopaminergic cell loss and dopamine-dependent behavioral deficits. This hurdle was overcome by direct targeting of the substantia nigra with viral vectors overexpressing PD-associated genes. Local gene delivery using viral vectors provides an attractive way to express transgenes in the central nervous system. Specific brain regions can be targeted (e.g. the substantia nigra), expression can be induced in the adult setting and high expression levels can be achieved. Further, different vector systems based on various viruses can be used. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based alpha-synuclein animal model for Parkinson&#39;s disease.

This manuscript describes how viral vector-mediated local gene delivery provides an attractive way to express transgenes in the central nervous system. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based animal model for Parkinson's disease.

Entwicklung eines Alpha-Synuklein Based Rattenmodell für Parkinson-Krankheit durch stereotaktische Injektion eines rekombinanten Adeno-assoziierten viralen Vektor,

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

Messung der isometrischen Kontraktilität der Maus mesenterialen Arterie mit Draht Myographie

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Acupuncture in a Rat Model of Asthma

A high platform can fix rats without restriction and completely expose the acupoints on the back during acupuncture manipulation. This article describes methods for the fabrication of the high platform, establishes a rat model of asthma and measures changes in respiratory function using a noninvasive and real-time whole-body plethysmography (WBP) system.

Akupunktur in einem Rattenmodell von Asthma

A high platform can fix rats without restriction and completely expose the acupoints on the back during acupuncture manipulation. This article describes ...

Retinal Pathophysiological Evaluation in a Rat Model

A posterior segment eye disease like diabetic retinopathy alters the physiology of the retina. Diabetic retinopathy is characterized by a retinal detachment, breakdown of the blood-retinal barrier (BRB), and retinal angiogenesis. An in vivo rat model is a valuable experimental tool to examine the changes in the structure and function of the retina. We propose three different experimental techniques in the rat model to identify morphological changes of retinal cells, retinal vasculature, and compromised BRB. Retinal histology is used to study the morphology of various retinal cells. Also, quantitative measurement is performed by retinal cell count and thickness measurement of different retinal layers. A BRB breakdown assay is used to determine the leakage of extraocular proteins from the plasma to vitreous tissue due to the breakdown of BRB. Fluorescence angiography is used to study angiogenesis and leakage of blood vessels by visualizing retinal vasculature using FITC-dextran dye.

Diabetic retinopathy is one of the leading causes of blindness. Histology, blood-retinal barrier breakdown assay, and fluorescence angiography are valuable techniques to understand the pathophysiology of the retina, which could further enhance the efficient drug screening against diabetic retinopathy.

Retinale pathophysiologische Bewertung in einem Rattenmodell

A posterior segment eye disease like diabetic retinopathy alters the physiology of the retina. Diabetic retinopathy is characterized by a retinal ...

Development of a Uterosacral Ligament Suspension Rat Model

Pelvic organ prolapse (POP) is a common pelvic floor disorder (PFD) with the potential to significantly impact a woman's quality of life. Approximately 10%-20% of women undergo pelvic floor repair surgery to treat prolapse in the United States. PFD cases result in an overall $26.3 billion annual cost in the United States alone. This multifactorial condition has a negative impact on the quality of life and yet the treatment options have only dwindled in the recent past. One common surgical option is uterosacral ligament suspension (USLS), which is typically performed by affixing the vaginal vault to the uterosacral ligament in the pelvis. This repair has a lower incidence of complications compared to those with mesh augmentation, but is notable for a relatively high failure rate of up to 40%. Considering the lack of standard animal models to study pelvic floor dysfunction, there is an urgent clinical need for innovation in this field with a focus on developing cost-effective and accessible animal models. In this manuscript, we describe a rat model of USLS involving a complete hysterectomy followed by fixation of the remaining vaginal vault to the uterosacral ligament. The goal of this model is to mimic the procedure performed on women to be able to use the model to then investigate reparative strategies that improve the mechanical integrity of the ligament attachment. Importantly, we also describe the development of an in situ tensile testing procedure to characterize interface integrity at chosen time points following surgical intervention. Overall, this model will be a useful tool for future studies that investigate treatment options for POP repair via USLS.

Pelvic organ prolapse affects millions of women worldwide and yet some common surgical interventions have failure rates as high as 40%. The lack of standard animal models to investigate this condition impedes progress. We propose the following protocol as a model for uterosacral ligament suspension and in vivo tensile testing.

Entwicklung eines uterosakralen Ligament-Suspensionsrattenmodells

Pelvic organ prolapse (POP) is a common pelvic floor disorder (PFD) with the potential to significantly impact a woman's quality of life. Approximately ...

A Model of Long-Term Ventricular Fibrillation in Isolated Rat Hearts

Ventricular fibrillation (VF) is a fatal arrhythmia with a high incidence in cardiac patients, but VF arrest under perfusion is a neglected method of intraoperative arrest in the field of cardiac surgery. With recent advances in cardiac surgery, the demand for prolonged VF studies under perfusion has increased. However, the field lacks simple, reliable, and reproducible animal models of chronic ventricular fibrillation. This protocol induces long-term VF through alternating current (AC) electrical stimulation of the epicardium. Different conditions were used to induce VF, including continuous stimulation with a low or high voltage to induce long-term VF and stimulation for 5 min with a low or high voltage to induce spontaneous long-term VF. The success rates of the different conditions, as well as the rates of myocardial injury and recovery of cardiac function, were compared. The results showed that continuous low-voltage stimulation induced long-term VF and that 5 min of low-voltage stimulation induced spontaneous long-term VF with mild myocardial injury and a high rate of recovery of cardiac function. However, the low-voltage, continuously stimulated long-term VF model had a higher success rate. High-voltage stimulation provided a higher rate of VF induction but showed a low defibrillation success rate, poor recovery of cardiac function, and severe myocardial injury. On the basis of these results, continuous low-voltage epicardial AC stimulation is recommended for its high success rate, stability, reliability, reproducibility, low impact on cardiac function, and mild myocardial injury.

This protocol presents a model of long-term ventricular fibrillation in rat hearts induced by continuous stimulation with low-voltage alternating current. This model has a high success rate, is stable, reliable, and reproducible, has a low impact on cardiac function, and causes only mild myocardial injury.

Ein Modell für Langzeit-Kammerflimmern in isolierten Rattenherzen

Ventricular fibrillation (VF) is a fatal arrhythmia with a high incidence in cardiac patients, but VF arrest under perfusion is a neglected method of ...