Diese Arbeit wurde durch die Cork South Infirmary Victoria University Hospital Breast Fonds, die Irish Cancer Society (CRI07TAN) und Cork Krebsforschungszentrum finanziert. Lewis Lungenkrebs-Zelllinie stabil exprimieren Luciferase war eine Art Geschenk von Dr. Karin Jooss, Cell Genesys, Inc., San Francisco, USA.

<html> Einführung Die Leber ist oft der erste Ort der Metastasen und kann der einzige Ort der Ausbreitung in nicht weniger als 30 40% der Patienten mit fortgeschrittener Erkrankung sein. Allerdings Optionen zur Behandlung von metastasierenden Tumoren der Leber sind nur wenige mit einer Minderheit der Patienten für Resektion und nur 23% größeren Responder Chemotherapeutika. Präklinische Forschung in diesem Bereich durch das Fehlen eines geeigneten Mausmodell zu isolieren Lebererkrankung Studie beschränkt. Wir beschreiben eine Split-Milz-Ansatz, mit dem zuverlässig einheitliche, diffuse Lebermetastasen werden kann. Resektion von dieser Hälfte der Milz verhindert die Folgen der Milztumor Wachstum und die andere Hälfte der Milz ist mit einem intakten Pfortaderkreislauf zur Milz Immunsystem schützenden Eigenschaften halten zurückgegeben. Das Modell, das wir beschreiben, ist ziemlich einfach durchzuführen und kann schnell durch einen einzigen Schnitt gemacht werden. Die Biolumineszenz Eigenschaften des Tumor-Zelllinie erlaubt eine genaue Quantifizierung Ansprechen auf die Behandlung ohne Tieropfer. Protokoll I. Cell Preparation <ol><li> Lewis Lungenkarzinom-Zellen sind Dulbecco Modified Essential Medium mit 10% fötalem Kälberserum bei 37 ergänzt ° C, 5% CO 2 angebaut und geerntet durch Trypsinierung bei sub-konfluenten. </li><li> Die Zellen wurden gezählt mit einem NucleoCounter (Chemo-metec, Dänemark) und eine einzelne Zelle Suspension von 1 x 10 6 Zellen in 200 ul wurde in serumfreien Medien vorbereitet. </li></ol> II. Maus Vorbereitung Hinweis: Alle Arbeiten wurden von der Ethikkommission des University College Cork genehmigt. In unserem Labor verwenden wir Injektionsnarkosemittel, um die Maus zu betäuben, alternativ kann man Inhalationsanästhetika zu verwenden, um denselben Effekt zu erzielen. Die Maus-Stamm verwendeten wir die haarlose MF1 nu / nu-Stamm von Harlan Laboratories (Oxfordshire, England) gewonnen. Weibliche MF1 nu / nu Mäuse mit einem Gewicht 16-22 g und von 6-8 Wochen alt waren in diesem Experiment einbezogen. Andere Stämme wie die C57-und Balb / c kann auch mit geeigneten Tumor-Zelllinien verwendet werden. <ol><li> Anästhesie wurde durch intraperitoneale Injektion von 100 ul von 1,5 mg Ketamin und 300 ug Xylazin geliefert. </li><li> Die Narkosetiefe wird anhand Zehen klemmen. Es sollte kein Rückzug Reflex mit toe Prise werden. </li><li> Die narkotisierten Maus richtig positioniert ist und mit Klebeband über ein Heizkissen. </li><li> Der Bauch ist mit einem 10% betadine Lösung vorbereitet. </li><li> Die Bauch-und OP-Gebiet sind in einer sterilen Mode drapiert. </li></ol> III. Bauchschnitt <ol><li> Eine kleine subkostalen Einschnitt in die Haut gemacht. </li><li> Der Schnitt wird um 2-3 cm verlängert. </li><li> Die Bauchwandmuskulatur wurden geteilt und Bauchhöhle getreten. </li></ol> IV. Belichtung und Division of Spleen <ol><li> Die Milz ist mobilisiert und sanft exteriorisierten auf einem vorgestanzten steriler Gaze. </li><li> Zwei 4-0 Vicryl Ligaturen um die Mitte des Körpers der Milz gebunden. </li><li> Milz ist zwischen den beiden Ligaturen die Schaffung von zwei hemi-Milzen unterteilt. </li></ol> V. Injektion von Zellen in die Hemi-Milz <ol><li> A 27 G wird benutzt, um eine 100-ul-Volumen von Zellen in einem Hemi-Milz zu injizieren. </li><li> Tumorzellen sind zulässig, um die Leber für 10 Minuten eine ausreichende Zeit für die Mehrheit der injizierten Zellen in die Leber gelangen abtropfen lassen. </li></ol> VI. Exzision von Hemi-Milz-, Viszeral-Verschluss und Wiederherstellung <ol><li> Die injizierten Hemi-Milz herausgeschnitten wird. </li><li> Die anderen Hemi-Milz ist in die Bauchhöhle zurück. </li><li> Die Muskelschicht ist mit 4-0 PDS Naht mit einem einfachen Laufstich geschlossen. </li><li> Die Bauchhaut wird mit 4-0 Prolene mit unterbrochenen Nähten verschlossen. </li><li> Fluid-Bolus verabreicht und postoperativen Analgesie, Carpofen (5 mg / kg) ist durch ip-Injektion für 24 h nach der Operation verabreicht wird. </li><li> Die Maus ist erlaubt, aus der Narkose erholen </li></ol> VII. IVIS Whole Body Imaging Bioluminescent <ol><li> Die Maus ist betäubt mit injizierbaren Betäubung. </li><li> Luciferin-Substrat wird durch intra-peritoneale Injektion 10 Minuten vor dem Imaging gegeben </li><li> Mäuse sind auf der Bühne platziert und bebildert für 3 min mit IVIS Ganzkörper-Imaging-System </li><li> Lumineszenz aus der Leber lassen sich mit einem region of interest-Tool werden </li></ol> VIII. Die Exposition von Mausleber <ol><li> Die Maus ist betäubt mit injizierbaren Betäubung. </li><li> Ein Dach Bauchschnitt ermöglicht die Visualisierung der diffuse Lebermetastasen in vivo. </li><li> Die Maus ist durch Genickbruch getötet und die Leber kann zur weiteren Analyse ausgeschnitten werden. </li></ol>_upload/1977/1977fig1.jpg &quot;alt =&quot; Bild 1 &quot;/&gt; Abbildung 1. Frisch herausgeschnitten murine unbehandelt und Tumor-infiltrierte Leber. <img src="/files/ftp_upload/1977/1977fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Kinetik der Leber Tumorwachstum durch IVIS Ganzkörperbildgebungssysteme belegt.

<ol>
	<li>Kasuya, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+subcutaneous+spleen+reservoir+for+multiple+portal+venous+injections+to+treat+liver+malignancies.">Mouse models of subcutaneous spleen reservoir for multiple portal venous injections to treat liver malignancies.</a> Cancer Res. 65, 3823-3827 (2005).</li><li>Liu, Q. Z., Tuo, C. W., Wang, B., Wu, B. Q., Zhang, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+metastasis+models+of+human+colorectal+carcinoma+established+in+nude+mice+by+orthotopic+transplantation+and+their+biologic+characteristic.">Liver metastasis models of human colorectal carcinoma established in nude mice by orthotopic transplantation and their biologic characteristic.</a> World J Gastroenterol. 4, 409-411 (1998).</li><li>Li, Z. Y., Ni, S., Yang, X., Kiviat, N., Lieber, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenograft+models+for+liver+metastasis:+Relationship+between+tumor+morphology+and+adenovirus+vector+transduction.">Xenograft models for liver metastasis: Relationship between tumor morphology and adenovirus vector transduction.</a> Mol Ther. 9, 650-657 (2004).</li><li>Cashman, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+gene+therapy+as+a+neoadjuvant+to+surgical+excision+to+control+metastatic+cancers.">Immune gene therapy as a neoadjuvant to surgical excision to control metastatic cancers.</a> Cancer Lett. , (2008).</li></ol>

Die Entwicklung von In-vivo-Modelle für die Untersuchung von Lebermetastasen wird immer steigt mit dem Aufkommen von neuen therapeutischen Strategien wichtig. Für die genaue Untersuchung von neuen Strategien, Tiermodelle sollte relativ einfach durchzuführen, bilden eine bedeutende Anzahl von Metastasen, beinhalten alle Schritte der metastatischen Kaskade und ein Reporter-System, das Erfassen und Zählen von einer begrenzten Anzahl von metastatischen Zellen ermöglicht. Die Split-Milz-Ansatz wie beschrieben über einen einzigen Betrieb und ist eine relativ einfache Technik zu erlernen, um eine einheitliche diffuse Lebermetastasen mit einem sehr geringen Risiko von Karzinomatose und Pfortaderthrombose 1 zu begründen. Die Aufteilung der Milz in zwei Hemi-Milzen ermöglichen die Injektion von Tumorzellen in einem Hemi-Milz. Tumorzellen geben den Pfortaderkreislauf und Reisen in die Leber, imitiert den ersten Schritt der metastatischen Kaskade. Excision der injizierten Hemi-Milz verhindert das Wachstum eines Milztumor und die Rückkehr der anderen Hemi-Milz sorgt für die Beibehaltung der Milz immunologischen Eigenschaften, die das Tier hatte vor dem Eingriff .. Dieses Modell kann für eine Vielzahl von Tumorarten eingesetzt werden. Die Lewis-Lungen-Luciferase-Zelllinie ermöglicht die Biolumineszenz Quantifizierung von Lebererkrankungen im Laufe der Zeit. Die erhöhte Empfindlichkeit der Biolumineszenz-System ermöglicht die Detektion und Quantifizierung von einer kleinen Anzahl von metastatischen Zellen. Dies ermöglicht eine genaue Zeitpunkt für die Einleitung der Behandlung Protokolle und ermöglicht die Quantifizierung der Lebererkrankung ohne Opfer des Tieres. Die Einfachheit, Haltbarkeit und Reproduzierbarkeit machen dieses Modell geeignet für die Untersuchung der verschiedenen Behandlungsstrategien einschließlich neuartiger Chemotherapeutika 2, Gentherapie 3 und Immuntherapie 1,4.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>D6429</td>
<td>&nbsp;</td>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>D8537</td>
<td>&nbsp;</td>
<tr>
<td>Ketamine</td>
<td>&nbsp;</td>
<td>Vetoquinol</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Xylazine</td>
<td>&nbsp;</td>
<td>Vetoquinol</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Carprofen</td>
<td>&nbsp;</td>
<td>Vetoquinol</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Videne</td>
<td>&nbsp;</td>
<td>Ecolab, Leeds, UK</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Vicryl 4-0 suture</td>
<td>&nbsp;</td>
<td>Ethicon</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>PDS 4-0 suture</td>
<td>&nbsp;</td>
<td>Ethicon</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Prolene 4-0 suture</td>
<td>&nbsp;</td>
<td>Ethicon</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Firefly Luciferin</td>
<td>&nbsp;</td>
<td>Biosynth, Zurich, Switzerland</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

murine bioluminescent hepatic tumour model

Dieses Video beschreibt die Einrichtung von Lebermetastasen in einem Mausmodell, die anschließend durch Biolumineszenz Imaging analysiert werden können. Tumorzellen sind speziell auf die Leber, um eine lokalisierte Lebertumor induzieren, über die Mobilisierung der Milz und eine Aufspaltung in zwei, intakt gelassen den Gefäßstiel für jede Hälfte der Milz. Lewis Lungenkarzinom-Zellen, die konstitutiv das Luciferase-Gen (luc1) sind in einem Hemi-Milz, die dann reseziert wird 10 Minuten später geimpft. Die anderen Hemi-Milz ist intakt gelassen und kehrte auf den Bauch. Liver Tumorwachstum kann durch Biolumineszenz-Bildgebung mit dem IVIS Ganzkörperbildgebungssysteme System überwacht werden. Quantitative Bildgebung des Tumorwachstums mit IVIS bietet eine präzise Quantifizierung der vitalen Tumorzellen. Tumor Zelltod und Nekrose durch medikamentöse Behandlung ist eine frühzeitige durch eine Reduktion der Biolumineszenz-Signal angezeigt. Dieses Mausmodell ermöglicht die Untersuchung der zugrunde liegenden Mechanismen metastasierten Tumor-Zell-Überleben und Wachstum und kann für die Evaluierung von Therapeutika von Lebermetastasen eingesetzt werden.

Watch this Scientific Journal Video about Murine Bioluminescent Hepatic Tumour Model at JoVE.com

Murine Bioluminescent Hepatic Tumour Model

Dieser Artikel beschreibt ein Verfahren zur Induktion der orthotopen Biolumineszenz Lebertumore bei Mäusen, und die anschließende Analyse des Tumorwachstums beschränkt sich auf die Leber mit Live-Ganzkörper-Lumineszenz-Imaging.

Murine Bioluminescent Leber Tumor-Modell

This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are ...

murine-bioluminescent-hepatic-tumour-model

Nanyang Technological University

Research

JoVE Journal

Medicine

17.1K Aufrufe.  University College Cork. Dieser Artikel beschreibt ein Verfahren zur Induktion der orthotopen Biolumineszenz Lebertumore bei Mäusen, und die anschließende Analyse des Tumorwachstums beschränkt sich auf die Leber mit Live-Ganzkörper-Lumineszenz-Imaging.

Serie: Murine Bioluminescent Hepatic Tumour Model

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes 1-3.
The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of &alpha;-smooth muscle actin (&alpha;-SMA) expression4,5. These cells are a hallmark of 'activated fibroblasts' that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues 6. The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients.
Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis 7-10. CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model.
In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance 11.
We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.

Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

Carcinoma-associated fibroblasts (CAFs) rich in myofibroblasts present within the tumour stroma, play a major role in driving tumour progression. We developed a coimplantation tumour xengraft model for experimentally generating CAFs from human mammary fibroblasts. The protocol describes how to establish CAF myofibroblasts that acquire an ability to promote tumourigenesis.

Experimentelle Erzeugung von Karzinom-assoziierte Fibroblasten (CAF) von menschlichen Brustkrebszellen Fibroblasten

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of ...

Forschung

Medizin

In vivo Dual Substrate Bioluminescent Imaging

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate since the advent of in vivo bioluminescent imaging technologies 1-3. Indeed, the ability to locate and quantify tumor growth longitudinally in a single cohort of animals to completion of the study as opposed to sacrificing individual groups of animals at specific assay times has revolutionized how researchers investigate breast cancer metastasis. Unfortunately, current methodologies preclude the real-time assessment of critical changes that transpire in cell signaling systems as breast cancer cells (i) evolve within primary tumors, (ii) disseminate throughout the body, and (iii) reinitiate proliferative programs at sites of a metastatic lesion. However, recent advancements in bioluminescent imaging now make it possible to simultaneously quantify specific spatiotemporal changes in gene expression as a function of tumor development and metastatic progression via the use of dual substrate luminescence reactions. To do so, researchers take advantage for two light-producing luciferase enzymes isolated from the firefly (Photinus pyralis) and sea pansy (Renilla reniformis), both of which react to mutually exclusive substrates that previously facilitated their wide-spread use in in vitro cell-based reporter gene assays 4. Here we demonstrate the in vivo utility of these two enzymes such that one luminescence reaction specifically marks the size and location of a developing tumor, while the second luminescent reaction serves as a means to visualize the activation status of specific signaling systems during distinct stages of tumor and metastasis development. Thus, the objectives of this study are two-fold. First, we will describe the steps necessary to construct dual bioluminescent reporter cell lines, as well as those needed to facilitate their use in visualizing the spatiotemporal regulation of gene expression during specific steps of the metastatic cascade. Using the 4T1 model of breast cancer metastasis, we show that the in vivo activity of a synthetic Smad Binding Element (SBE) promoter was decreased dramatically in pulmonary metastasis as compared to that measured in the primary tumor 4-6. Recently, breast cancer metastasis was shown to be regulated by changes within the primary tumor microenvironment and reactive stroma, including those occurring in fibroblasts and infiltrating immune cells 7-9. Thus, our second objective will be to demonstrate the utility of dual bioluminescent techniques in monitoring the growth and localization of two unique cell populations harbored within a single animal during breast cancer growth and metastasis.

In vivo Dual Substrate Bioluminescent Imaging

Herein we describe the methods to construct, visualize, and quantify the bioluminescent reactions of both firefly and renilla luciferase enzymes expressed in metastatic breast cancer cells during their growth and metastasis in vivo.

In vivo Dual-Substrat Bioluminescent Imaging

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate ...

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The cell line was originally induced by intracranial injection of 3-methyl-cholantrene into a C57BL/6 syngeneic mouse strain 1; therefore, immunologically competent C57BL/6 mice can be used. While we use GL261, the following protocol can be used for the implantation and monitoring of any intracranial mouse tumor model. GL261 cells were engineered to stably express firefly luciferase (GL261-luc). We also created the brighter GL261-luc2 cell line by stable transfection of the luc2 gene expressed from the CMV promoter. C57BL/6-cBrd/cBrd/Cr mice (albino variant of C57BL/6) from the National Cancer Institute, Frederick, MD were used to eliminate the light attenuation caused by black skin and fur. With the use of albino C57BL/6 mice; in vivo imaging using the IVIS Spectrum in vivo imaging system is possible from the day of implantation (Caliper Life Sciences, Hopkinton, MA). The GL261-luc and GL261-luc2 cell lines showed the same in vivo behavior as the parental GL261 cells. Some of the shared histological features present in human GBMs and this mouse model include: tumor necrosis, pseudopalisades, neovascularization, invasion, hypercellularity, and inflammation 1.
Prior to implantation animals were anesthetized by an intraperitoneal injection of ketamine (50 mg/kg), xylazine (5 mg/kg) and buprenorphine (0.05 mg/kg), placed in a stereotactic apparatus and an incision was made with a scalpel over the cranial midline. A burrhole was made 0.1mm posterior to the bregma and 2.3mm to the right of the midline. A needle was inserted to a depth of 3mm and withdrawn 0.4mm to a depth of 2.6mm. Two &mu;l of GL261-luc or GL261-luc2 cells (107 cells/ml) were infused over the course of 3 minutes. The burrhole was closed with bonewax and the incision was sutured.
Following stereotactic implantation the bioluminescent cells are detectable from the day of implantation and the tumor can be analyzed using the 3D image reconstruction feature of the IVIS Spectrum instrument. Animals receive a subcutaneous injection of 150&mu;g luciferin /kg body weight 20 min prior to imaging. Tumor burden is quantified using mean tumor bioluminescence over time. Tumor-bearing mice were observed daily to assess morbidity and were euthanized when one or more of the following symptoms are present: lethargy, failure to ambulate, hunched posture, failure to groom, anorexia resulting in &gt;10% loss of weight. Tumors were evident in all of the animals on necropsy.

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

Intracranial implantation of GL261 cells into C57BL/6 mice produces malignant gliomas that recapitulate many of the hallmarks of human glioblastoma multiforme. We used GL261 cells stably expressing luciferase to allow us to use in vivo imaging to follow tumor progression. The surgery and 3D in vivo imaging are demonstrated.

Intrakranielle Implantation mit anschließender 3D In Vivo Bioluminescent Imaging von Murine Gliome

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The ...

Surgical Procedures for a Rat Model of Partial Orthotopic Liver Transplantation with Hepatic Arterial Reconstruction

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as studies on graft preservation and ischemia-reperfusion injury 1,2, immunological responses 3,4, hemodynamics 5,6, and small-for-size syndrome 7. The rat OLT is among the most difficult animal models in experimental surgery and demands advanced microsurgical skills that take a long time to learn. Consequently, the use of this model has been limited. Since the reliability and reproducibility of results are key components of the experiments in which such complex animal models are used, it is essential for surgeons who are involved in rat OLT to be trained in well-standardized and sophisticated procedures for this model.
While various techniques and modifications of OLT in rats have been reported 8 since the first model was described by Lee et al. 9 in 1973, the elimination of the hepatic arterial reconstruction 10 and the introduction of the cuff anastomosis technique by Kamada et al. 11 were a major advancement in this model, because they simplified the reconstruction procedures to a great degree. In the model by Kamada et al., the hepatic rearterialization was also eliminated. Since rats could survive without hepatic arterial flow after liver transplantation, there was considerable controversy over the value of hepatic arterialization. However, the physiological superiority of the arterialized model has been increasingly acknowledged, especially in terms of preserving the bile duct system 8,12 and the liver integrity 8,13,14.
In this article, we present detailed surgical procedures for a rat model of OLT with hepatic arterial reconstruction using a 50% partial graft after ex vivo liver resection. The reconstruction procedures for each vessel and the bile duct are performed by the following methods: a 7-0 polypropylene continuous suture for the supra- and infrahepatic vena cava; a cuff technique for the portal vein; and a stent technique for the hepatic artery and the bile duct.

Orthotopic liver transplantation in rats is an indispensable experimental model for biomedical research. Here we present our surgical procedures for orthotopic rat liver transplantation with hepatic arterial reconstruction using a 50% partial graft.

Chirurgische Verfahren für eine Ratte Modell Teilweise Lebertransplantation mit arteriellen Rekonstruktion

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as ...

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.

Mausmodell der Wundheilung

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

Bioluminescent Orthotopic Model of Pancreatic Cancer Progression

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%. New therapeutic options are critically needed and depend on improved understanding of pancreatic cancer biology. To better understand the interaction of cancer cells with the pancreatic microenvironment, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression. Luciferase-tagged pancreatic cancer cells are resuspended in Matrigel and delivered into the pancreatic tail during laparotomy. Matrigel solidifies at body temperature to prevent leakage of cancer cells during injection. Primary tumor growth and metastasis to distant organs are monitored following injection of the luciferase substrate luciferin, using in vivo imaging of bioluminescence emission from the cancer cells. In vivo imaging also may be used to track primary tumor recurrence after resection. This orthotopic model is suited to both syngeneic and xenograft models and may be used in pre-clinical trials to investigate the impact of novel anti-cancer therapeutics on the growth of the primary pancreatic tumor and metastasis.

Improved understanding of pancreatic cancer biology is critically needed to enable the development of better therapeutic options to treat pancreatic cancer. To address this need, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression using in vivo bioluminescence imaging.

Bioluminescent orthotopen Modell von Bauchspeicheldrüsenkrebs Progression

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%.  New therapeutic options are critically needed and depend on improved ...

A Murine Model of Subarachnoid Hemorrhage

In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.

A standardized mouse model of subarachnoid hemorrhage by intraluminal Circle of Willis perforation is described. Vessel perforation and subarachnoid bleeding are monitored by intracranial pressure monitoring. In addition various vital parameters are recorded and controlled to maintain physiologic conditions.

Einem Mausmodell der Subarachnoidalblutung

In this video publication a standardized mouse model of subarachnoid  hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of  Willis ...

A Preclinical Murine Model of Hepatic Metastases

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, murine pancreatic tumor model of hepatic metastases via a hemispleen injection of syngeneic murine pancreatic tumor cells is described. This model mimics many of the clinical conditions in patients with metastatic disease to the liver. Mice consistently develop metastases in the liver allowing for investigation of the metastatic process, experimental therapy testing, and tumor immunology research.

A preclinical, murine model of hepatic metastases performed via a hemispleen injection technique.

Einer präklinischen Mausmodell der Lebermetastasen

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, ...

Murine Model of Intestinal Ischemia-reperfusion Injury

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, hypotension, necrotizing enterocolitis, bowel transplantation, trauma and chronic inflammation. Intestinal ischemia-reperfusion (IR) injury is a consequence of acute mesenteric ischemia, caused by inadequate blood flow through the mesenteric vessels, resulting in intestinal damage. Reperfusion following ischemia can further exacerbate damage of the intestine. The mechanisms of IR injury are complex and poorly understood. Therefore, experimental small animal models are critical for understanding the pathophysiology of IR injury and the development of novel therapies.
Here we describe a mouse model of acute intestinal IR injury that provides reproducible injury of the small intestine without mortality. This is achieved by inducing ischemia in the region of the distal ileum by temporally occluding the peripheral and terminal collateral branches of the superior mesenteric artery for 60 min using microvascular clips. Reperfusion for 1 hr, or 2 hr after injury results in reproducible injury of the intestine examined by histological analysis. Proper position of the microvascular clips is critical for the procedure. Therefore the video clip provides a detailed visual step-by-step description of this technique. This model of intestinal IR injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Here we describe the detailed procedure of intestinal ischemia-reperfusion in mice which results in reproducible injury without mortality to encourage the standardization of this technique across the field. This model of intestinal ischemia-reperfusion injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Murine Modell der intestinalen Ischämie-Reperfusionsschaden

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, ...

Method of Direct Segmental Intra-hepatic Delivery Using a Rat Liver Hilar Clamp Model

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ischemia/reperfusion (I/R) injury with myriad negative consequences. Potential I/R injury in marginal organs destined for liver transplantation contributes to the current donor shortage secondary to a decreased organ utilization rate. A significant need exists to explore hepatic I/R injury in order to mediate its impact on graft function in transplantation. Rat liver hilar clamp models are used to investigate the impact of different molecules on hepatic I/R injury. Depending on the model, these molecules have been delivered using inhalation, epidural infusion, intraperitoneal injection, intravenous administration or injection into the peripheral superior mesenteric vein. A rat liver hilar clamp model has been developed for use in studying the impact of pharmacologic molecules in ameliorating I/R injury. The described model for rat liver hilar clamp includes direct cannulation of the portal supply to the ischemic hepatic segment via a side branch of the portal vein, allowing for direct segmental hepatic delivery. Our approach is to induce ischemia in the left lateral and median lobes for 60 min, during which time the substance under study is infused. In this case, pegylated-superoxide dismutase (PEG-SOD), a free radical scavenger, is infused directly into the ischemic segment. This series of experiments demonstrates that infusion of PEG-SOD is protective against hepatic I/R injury. Advantages of this approach include direct injection of the molecule into the ischemic segment with consequent decrease in volume of distribution and reduction in systemic side effects.

A unique rat liver hilar clamp model was developed for studying the impact of pharmacologic molecules in ameliorating ischemia-reperfusion injury. This model includes direct cannulation of the portal supply to the ischemic liver segment via a branch of the portal vein, allowing for direct hepatic delivery.

Methode der direkten segmentale Intra-Leber-Lieferung Mit einer Rattenleber Hilar Clamp Modell

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ...