Diese Arbeit wird von der Goethe-Universität Frankfurt und einem Zuschuss von der Deutschen Forschungsgemeinschaft unterstützt, um UC

<ol>
	<li>Manns, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+and+management+of+autoimmune+hepatitis.">Diagnosis and management of autoimmune hepatitis.</a> Hepatology. 51, 2193-2213 (2010).</li><li>Czaja, A. J., Manns, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+the+Diagnosis,+Pathogenesis+and+Management+of+Autoimmune+Hepatitis.">Advances in the Diagnosis, Pathogenesis and Management of Autoimmune Hepatitis.</a> Gastroenterology. 4, 429-443 (2010).</li><li>Christen, U., Hintermann, E., Jaeckel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+animal+models+for+autoimmune+hepatitis.">New animal models for autoimmune hepatitis.</a> Semin. Liver Dis. 29, 262-272 (2009).</li><li>Christen, U., Holdener, M., Hintermann, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+for+autoimmune+hepatitis.">Animal models for autoimmune hepatitis.</a> Autoimmun. Rev. 6, 306-311 (2007).</li><li>Czaja, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+autoimmune+hepatitis.">Animal models of autoimmune hepatitis.</a> Expert Rev. Gastroenterol. Hepatol. 4, 429-443 (2010).</li><li>Holdener, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breaking+tolerance+to+the+natural+human+liver+autoantigen+cytochrome+P450+2D6+by+virus+infection.">Breaking tolerance to the natural human liver autoantigen cytochrome P450 2D6 by virus infection.</a> J. Exp. Med. 205, 1409-1422 (2008).</li><li>Manns, M. P., Griffin, K. J., Sullivan, K. F., Johnson, E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LKM-1+autoantibodies+recognize+a+short+linear+sequence+in+P450IID6,+a+cytochrome+P-450+monooxygenase.">LKM-1 autoantibodies recognize a short linear sequence in P450IID6, a cytochrome P-450 monooxygenase.</a> J. Clin. Invest. 88, 1370-1378 (1991).</li><li>Zanger, U. M., Hauri, H. P., Loeper, J., Homberg, J. C., Meyer, U. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibodies+against+human+cytochrome+P-450db1+in+autoimmune+hepatitis+type+II.">Antibodies against human cytochrome P-450db1 in autoimmune hepatitis type II.</a> Proc. Natl. Acad. Sci. U. S. A. 85, 8256-8260 (1988).</li><li>von Herrath, M. G., Fujinami, R. S., Whitton, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microorganisms+and+autoimmunity:+making+the+barren+field+fertile.">Microorganisms and autoimmunity: making the barren field fertile.</a> Nat. Rev. Microbiol. 1, 151-157 (2003).</li><li>Yardeni, T., Eckhaus, M., Morris, H. D., Huizing, M., Hoogstraten-Miller, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retro-orbital+injections+in+mice.">Retro-orbital injections in mice.</a> Lab. Anim. (NY). 40, 155-160 (2011).</li><li>Forbes, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morbidity+and+mortality+rates+associated+with+serial+bleeding+from+the+superficial+temporal+vein+in+mice.">Morbidity and mortality rates associated with serial bleeding from the superficial temporal vein in mice.</a> Lab. Anim. (NY). 39, 236-240 (2010).</li><li>Ehser, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mimicry+rather+than+identity+beaks+T+cell+tolerance+in+the+CYP2D6+mouse+model+for+human+autoimmune+hepatitis.">Molecular mimicry rather than identity beaks T cell tolerance in the CYP2D6 mouse model for human autoimmune hepatitis.</a> , (2011).</li><li>Hintermann, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epitope+spreading+of+the+anti-CYP2D6+antibody+response+in+patients+with+autoimmune+hepatitis+and+in+the+CYP2D6+mouse+model.">Epitope spreading of the anti-CYP2D6 antibody response in patients with autoimmune hepatitis and in the CYP2D6 mouse model.</a> J. Autoimmun. , (2011).</li><li>Ando, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Class+I-restricted+cytotoxic+T+lymphocytes+are+directly+cytopathic+for+their+target+cells+in+vivo.">Class I-restricted cytotoxic T lymphocytes are directly cytopathic for their target cells in vivo.</a> J. Immunol. 152, 3245-3253 (1994).</li><li>Zierden, M., Kuhnen, E., Odenthal, M., Dienes, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+and+regulation+of+autoreactive+CD8++T+cells+in+a+transgenic+mouse+model+of+autoimmune+hepatitis.">Effects and regulation of autoreactive CD8+ T cells in a transgenic mouse model of autoimmune hepatitis.</a> Gastroenterology. 139, 975-986 (2010).</li><li>Limmer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Failure+to+induce+organ-specific+autoimmunity+by+breaking+of+tolerance:+importance+of+the+microenvironment.">Failure to induce organ-specific autoimmunity by breaking of tolerance: importance of the microenvironment.</a> Eur. J. Immunol. 28, 2395-2406 (1998).</li><li>Voehringer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Break+of+T+cell+ignorance+to+a+viral+antigen+in+the+liver+induces+hepatitis.">Break of T cell ignorance to a viral antigen in the liver induces hepatitis.</a> J. Immunol. 165, 2415-2422 (2000).</li><li>Derkow, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+priming+of+CD8+and+CD4+T-cells+in+animal+models+of+autoimmune+hepatitis+and+cholangitis.">Differential priming of CD8 and CD4 T-cells in animal models of autoimmune hepatitis and cholangitis.</a> Hepatology. 46, 1155-1165 (2007).</li><li>Lapierre, P., Djilali-Saiah, I., Vitozzi, S., Alvarez, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+murine+model+of+type+2+autoimmune+hepatitis:+Xenoimmunization+with+human+antigens.">A murine model of type 2 autoimmune hepatitis: Xenoimmunization with human antigens.</a> Hepatology. 39, 1066-1074 (2004).</li><li>Djilali-Saiah, I., Lapierre, P., Vittozi, S., Alvarez, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+vaccination+breaks+tolerance+for+a+neo-self+antigen+in+liver:+a+transgenic+murine+model+of+autoimmune+hepatitis.">DNA vaccination breaks tolerance for a neo-self antigen in liver: a transgenic murine model of autoimmune hepatitis.</a> J. Immunol. 169, 4889-4896 (2002).</li><li>Christen, U., Hintermann, E., Holdener, M., von Herrath, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+triggers+for+autoimmunity:+is+the+'glass+of+molecular+mimicry'+half+full+or+half+empty.">Viral triggers for autoimmunity: is the 'glass of molecular mimicry' half full or half empty.</a> J. Autoimmun. 34, 38-44 (2010).</li><li>Krebs, P., Scandella, E., Odermatt, B., Ludewig, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+functional+exhaustion+and+deletion+of+CTL+following+immunization+with+recombinant+adenovirus.">Rapid functional exhaustion and deletion of CTL following immunization with recombinant adenovirus.</a> J. Immunol. 174, 4559-4566 (2005).</li><li>Hintermann, E., Bayer, M., Pfeilschifter, J., Christen, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenoviral+delivery+of+the+liver+autoantigen+cytochrome+P450+2D6+chronically+activates+hepatic+stellate+cells+and+induces+fibrosis.">Adenoviral delivery of the liver autoantigen cytochrome P450 2D6 chronically activates hepatic stellate cells and induces fibrosis.</a> Manuscript Submitted. , (2011).</li></ol>

Die Autoren haben nichts zu offenbaren.

In früheren Studien wurde experimentellen Hepatitis oft berichtet, dass sie nur vorübergehend und viele aktuelle Modelle für autoimmunen Lebererkrankungen auf einem ziemlich komplexe Erkrankung Induktion Protokolle (für Übersichten siehe 3,5) ab. Verwenden Sie z. B. mehrere Modelle transgenen Mäusen, die spezifische Ziel-Antigene und adoptiv übertragen Ziel-Antigen-spezifische, meist TcR-transgenen, T-Zellen 14,15. Oft eine zusätzliche Infektion mit livertropic Viren, Bakterien oder Parasiten ist nötig, Krankheit 16-18 zu induzieren. Alternativ wird die DNA-Impfung mit Plasmiden, die für die Ziel-Antigene, einschließlich CYP2D6 19, verwendet werden, um Hepatitis zu induzieren. Es wird jedoch ein zusätzliche Impfung mit Plasmiden, die für pro-inflammatorische Zytokine, wie IL-12, erforderlich 20. Leider mit einigen wenigen Ausnahmen 15,20 Hepatitis ist nur vorübergehend. Die CYP2D6-Modell 6,21 verwendet eine einfache Methode to induzieren Autoimmun-Hepatitis, indem Sie einfach Infektion von Mäusen mit einem Adenovirus, hCYP2D6, dem wichtigsten Autoantigen in AIH-2 7,8. Lieferung von CYP2D6 durch einen Adenovirus Konstrukt garantiert sowohl eine direkte Ausrichtung der Leber sowie eine lokale Entzündung, die den Abbau von Toleranz fördert. Es ist jedoch wichtig darauf hinzuweisen, dass sowohl der Virustiter sowie die Art der Verabreichung von entscheidender Bedeutung für dieses Modell an ist. Auf der einen Seite, ist Ad-2D6 eine Replikation defizienten Virus, damit, eine ausreichend hohe Titer von Virus benötigt, um eine kritische Menge an Antigen in der Leber zu erzeugen. Auf der anderen Seite könnte eine zu hohe Titer in einem fatalen akutem Leberversagen führen. Darüber hinaus hat es sich bereits erwiesen, dass Infektion von Mäusen durch hohe Titer von Adenovirus auf eine funktioneller Erschöpfung der Immunantwort 22 führt demonstriert. In unserem Modell haben wir eine Kombination von intravenöser und intraperitonealer Infektion, um sowohl die chronische zelluläre Infiltration sowie ext erhaltenensive Fibrose. Wir haben beobachtet, dass die intravenöse Infektion allein noch verursacht massive zelluläre Infiltration, aber keine Fibrose der subkapsulären Region, was darauf hinweist, dass die peritoneale Entzündung aufgrund der intraperitonealen Injektion könnte in der kontinuierlichen subkapsulären Ansammlung von Kollagen (Hintermann &amp; Christen, Manuskript in Vorbereitung) beteiligt sein. Es ist jedoch wichtig zu beachten, dass die beobachtete Leberfibrose Antigen-spezifische ist, da wir nicht erkennen Aktivierung von hepatischen Sternzellen und Ablagerung von Kollagen nach der Infektion mit dem gleichen Virus-Titer von Ad-GFP-oder Ad-Control 23. Die CYP2D6-Modell spiegelt viele Aspekte des menschlichen AIH 1,2, wie chronische Hepatitis durch anhaltende zelluläre Infiltration und Leberfibrose 6 gekennzeichnet. Darüber hinaus zeigt das Vorhandensein von hohem Titer Anti-CYP2D6 Antikörper mit ähnlichen Epitop-Spezifität und Verbreitung 13 bis LKM-1-Antikörper bei der AIH-2-Patienten fanden die Presence einer gemeinsamen chronische Autoimmun-Reaktivität. CYP2D6 ist die wichtigste Antigen in AIH-2, aber es wird nicht von Patienten mit der häufigeren Typ 1 AIH diagnostiziert anerkannt. Darüber hinaus Patienten mit anderen Lebererkrankungen mit autoimmunen Ätiologie, wie der primär biliären Zirrhose (PBC) oder primär sklerosierende Cholangitis (PSC), erzeugen Markenzeichen Autoantikörper mit Spezifität für verschiedene Autoantigene Leber und ihre Lebern zeigen verschiedene pathologische Merkmale Zentrierung auf kleine (PBC ) oder große (PSC) Gallengänge. Dennoch bietet die CYP2D6-Modelle die Möglichkeit, immunpathogenen Mechanismen der chronischen hepatischen entzündlichen Prozessen wie auftretenden autoimmunen Lebererkrankungen, zu wichtigen Akteuren, die die autoimmune Zerstörung der Hepatozyten zu fahren und eine mögliche therapeutische Interventionen zu bewerten, um die Krankheit zu heilen identifizieren beteiligt zu analysieren.

<table border="1"><tbody><tr><td> Bezeichnung des Geräts </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> 23G Nadel </td><td> BD Biosciences </td><td> 300800 </td><td></td></tr><tr><td> 27 ½ G Nadel </td><td> VWR </td><td> 612-0151 </td><td></td></tr><tr><td> 30 ½ G Nadel </td><td> BD Biosciences </td><td> 304000 </td><td></td></tr><tr><td> Alanin-Aminotransferase-Teststreifen (GPT / ALT) </td><td> Roche Diagnostics </td><td> 10 745 138 202 </td><td></td></tr><tr><td> Aspartat-Aminotransferase-Teststreifen (GOT / AST) </td><td> Roche Diagnostics </td><td> 10 745 120 202 </td><td></td></tr><tr><td> Anästhesiegerät Univentor 400 </td><td> <a href="http://www.agnthos.se" target="_blank" >AgnTho die</a> </td><td></td><td></td></tr><tr><td> Bodenformen, Einweg 37x24x10 mm </td><td> VWR </td><td> 720-0208 </td><td></td></tr><tr><td> Zellsieb 70mm </td><td> VWR </td><td> 734-0003 </td><td></td></tr><tr><td> Kryostat </td><td> Leica </td><td> CM1850 UV </td><td></td></tr><tr><td> Heparinisiertes Kapillarröhren </td><td> Fischer </td><td> 3123987 </td><td></td></tr><tr><td> Objektträger Superfrost Plus </td><td> Menzel Gläser </td><td> J1800AMNZ </td><td></td></tr><tr><td> Microtainer SST Rohre </td><td> BD Biosciences </td><td> 365951 </td><td></td></tr><tr><td> Mikrotiterplatten, V-Boden </td><td> VWR </td><td> 391-1924 </td><td></td></tr><tr><td> Reflotron Plus </td><td> Roche Diagnostics </td><td></td><td> Determiniation des Serum-AST / ALT </td></tr><tr><td colspan="4"></td></tr><tr><td> O Namef das Reagenz </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> Kaninchen anti-Maus-Anti-Kollagen Typ I-IgG-, </td><td> Chemicon </td><td> AB765P </td><td></td></tr><tr><td> Biotinylierte Ziegen-Anti-Kaninchen-IgG </td><td> Vector (AXXORA) </td><td> VC-BA-1000-MC15 </td><td></td></tr><tr><td> FITC-konjugiertem Anti-Maus-CD8a antibody </td><td> Southern Biotech </td><td> 1550-02 </td><td></td></tr><tr><td> PE-konjugierten anti-Maus-IFN &amp;ggr; antibody </td><td> BD Biosciences </td><td> 554412 </td><td></td></tr><tr><td> PE-konjugiertem anti-Maus-CD16/CD32 antibody (FcR Block) </td><td> BD Biosciences </td><td> 553141 </td><td></td></tr><tr><td> Aquatex </td><td> VWR </td><td> 1.08562.0050 </td><td></td></tr><tr><td> Avidin / Biotin-Blocking-Kit </td><td&gt; Vector (AXXORA) </td><td> VC-SP-2001-KI01 </td><td></td></tr><tr><td> Brefeldin A </td><td> Sigma </td><td> B6542 </td><td></td></tr><tr><td> Collagenase IV </td><td> Sigma </td><td> C5138-100MG </td><td></td></tr><tr><td> DAB Substrat Kit 3&#39;3&#39;diaminobenzidine </td><td> Vector (AXXORA) </td><td> VC-SK-4100-KI01 </td><td></td></tr><tr><td> DNase </td><td> Sigma </td><td> DN-25 </td><td></td></tr><tr><td> Elite ABC-Reagenz </td><td> Vector (AXXORA) </td><td> VC-PK-7100-L050 </td><td></td></tr><tr><td> Eosin G / Y-Lösung </td><td> Roth </td><td> X883.1 </td><td></td></tr><tr><td> Fötales Kälberserum (FCS) </td><td> Biochrom AG </td><td> S 0115 </td><td></td></tr><tr><td> Isofluran </td><td> Forene </td><td> B506 </td><td></td></tr><tr><td> Meyers Hämatoxylin-Lösung </td><td> AppliChem </td><td> A4840, 1000 </td><td></td></tr><tr><td> OCT-Verbindung </td><td> Sakura Finetek Europa </td><td> 4583 </td><td></td></tr><tr><td> PBS-gepuffertem Formaldehyd 10% </td><td> Roth </td><td> A146.3 </td><td></td></tr><tr><td> Percoll </td><td> Amersham </td><td> 17-0891-01 </td><td></td></tr><tr><td> Roti-Histokit </td><td> Roth </td><td> 6638,1 </td><td></td></tr><tr><td> RPMI </td><td> Invitrogen </td><td> 61870 </td><td></td></tr><tr><td> Saponin aus Quillaja Rinde </td><td> Sigma </td><td> S7900 </td><td></td></tr></tbody></table>

the cyp2d6 animal model: how to induce autoimmune hepatitis in mice

Autoimmun-Hepatitis ist eine seltene, aber lebensbedrohliche Autoimmunerkrankung der Leber unbekannter Ätiologie 1,2. In der Vergangenheit viele Versuche unternommen worden, es, ein Tiermodell, die die Eigenschaften der humanen Erkrankung 3-5 spiegelt zu erzeugen. Allerdings in verschiedenen Modellen die Induktion der Erkrankung war sehr komplex und oft nur vorübergehend war, Hepatitis 3-5. Deshalb haben wir einen einfachen Maus-Modell, das die wesentlichen menschlichen Autoantigen verwendet bei Typ-2 Autoimmunhepatitis (AIH-2), nämlich hCYP2D6, als Auslöser 6 entwickelt. Typ-1-Leber-Niere-Mikrosomen-Antikörper (LKM-1) Antikörper, die hCYP2D6 sind das Markenzeichen der AIH-2 7,8. Lieferung von hCYP2D6 in Wildtyp FVB oder C57BL / 6 Mäusen wurde von einem Adenovirus Konstrukt (Ad-2D6), die einer direkten Lieferung des auslösenden Antigens an der Leber sorgt. Somit erzeugt die daraus resultierende lokale Entzündung ein fruchtbares Feld 9 für die spätere Entwicklung von Autoimmunität. A combination von intravenöser und intraperitonealer Injektion von Ad-2D6 ist der effektivste Weg, um eine lang anhaltende autoimmunen Schädigungen der Leber (Abschnitt 1) ​​zu induzieren. Hier bieten wir Ihnen ein ausführliches Protokoll, wie autoimmunen Lebererkrankungen wird in der CYP2D6-Modell induziert und wie die verschiedenen Aspekte von Leberschäden beurteilt werden kann. Zunächst werden die Serumspiegel von Markierungen, die Hepatozyten-Zerstörung, wie Aminotransferasen, sowie die Titer von Antikörpern durch hCYP2D6 Blutentnahme retroorbitaly (Abschnitt 2) bestimmt. Zweitens wird die hCYP2D6-spezifischen T-Zell-Antwort durch das Sammeln von Lymphozyten aus der Milz und der Leber dadurch gekennzeichnet. Um reines Leber-Lymphozyten zu erhalten, werden die Lebern von PBS über die Pfortader (Abschnitt 3), in Kollagen verdaut und gereinigt über einen Percoll-Gradienten (Abschnitt 4) perfundiert. Die Häufigkeit der hCYP2D6-spezifischen T-Zellen wird durch Stimulation mit hCYP2D6 Peptide und Identifizierung von IFN-produzierenden Zellen mittels Durchflusszytometrie (Abschnitt 5) analysiert. Drittenszelluläre Infiltration und Fibrose wird durch Immunhistochemie von Leber-Abschnitte (Abschnitt 6) bestimmt. Eine solche Analyse Regime muss zu mehreren Zeitpunkten nach Beginn der Erkrankung durchgeführt werden, um die chronische Natur des Modells zu beweisen. Die Größe der Immunantwort durch die Frequenz und Aktivität von hCYP2D6-spezifischen T-und / oder B-Zellen und dem Grad der Leberschädigung und Fibrose dadurch gekennzeichnet haben, um für eine spätere Auswertung der möglichen Behandlungen zu verhindern, verzögern oder aufheben Autodestruktiven beurteilt werden Verfahren nach der Leber.

Watch this Scientific Journal Video about The CYP2D6 Animal Model: How to Induce Autoimmune Hepatitis in Mice at JoVE.com

The CYP2D6 Animal Model: How to Induce Autoimmune Hepatitis in Mice

Infektion von Mäusen mit einem Adenovirus, welches das wesentliche humane Autoantigen Cytochrom P450 2D6 (hCYP2D6) durch Seren von Patienten mit Typ-2-Autoimmun-Hepatitis führt zu einer persistierenden Form der Autoimmun-vermittelten Lebererkrankung, die durch umfangreiche Hepatitis, Fibrose und Erzeugung eines CYP2D6 gekennzeichnet anerkannt -spezifische Immunantwort.

Die CYP2D6-Tiermodell: Wie Autoimmune Hepatitis in Mäusen zu induzieren

Autoimmune hepatitis is a rare but life threatening autoimmune disease of the liver of unknown etiology1,2. In the past many attempts have been made to ...

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18.9K Aufrufe.  Goethe University Hospital Frankfurt. Infektion von Mäusen mit einem Adenovirus, welches das wesentliche humane Autoantigen Cytochrom P450 2D6 (hCYP2D6) durch Seren von Patienten mit Typ-2-Autoimmun-Hepatitis führt zu einer persistierenden Form der Autoimmun-vermittelten Lebererkrankung, die durch umfangreiche Hepatitis, Fibrose und Erzeugung eines CYP2D6 gekennzeichnet anerkannt -spezifische Immunantwort.

Serie: The CYP2D6 Animal Model: How to Induce Autoimmune Hepatitis in Mice

Pressure Controlled Ventilation to Induce Acute Lung Injury in Mice

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged mechanical ventilation, contributes to morbidity and mortality of critical illness, and studies on novel genetic or pharmacological targets are areas of intense investigation (1-3, 5, 8, 26, 30, 33-36). ALI is defined by the acute onset of the disease, which leads to non-cardiac pulmonary edema and subsequent impairment of pulmonary gas exchange (36). We have developed a murine model of ALI by using a pressure-controlled ventilation to induce ventilator-induced lung injury (2). For this purpose, C57BL/6 mice are anesthetized and a tracheotomy is performed followed by induction of ALI via mechanical ventilation. Mice are ventilated in a pressure-controlled setting with an inspiratory peak pressure of 45 mbar over 1 - 3 hours. As outcome parameters, pulmonary edema (wet-to-dry ratio), bronchoalveolar fluid albumin content, bronchoalveolar fluid and pulmonary tissue myeloperoxidase content and pulmonary gas exchange are assessed (2). Using this technique we could show that it sufficiently induces acute lung inflammation and can distinguish between different treatment groups or genotypes (1-3, 5). Therefore this technique may be helpful for researchers who pursue molecular mechanisms involved in ALI using a genetic approach in mice with gene-targeted deletion.

A murine model for ventilator induced lung injury is an important tool to study an acute lung injury in vivo. Here, we report an easy applicable in situ model for acute lung injury using high-pressure mechanical ventilation to induce acute failure of the lung.

Pressure Controlled Ventilation zu Acute Lung Injury in Mäusen zu induzieren

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged ...

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Medizin

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Biomarker in einem Tiermodell für die Aufdeckung Neural, hämatologischen und Behavioral Korrelate der PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Protocol for Long Duration Whole Body Hyperthermia in Mice

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body temperature that is observed during fever. The use of hyperthermia as an adjuvant has emerged as a promising procedure for tumor regression in the field of cancer biology. For this purpose, the most important requirement is to have reliable and uniform heating protocols. We have developed a protocol for hyperthermia (whole body) in mice. In this protocol, animals are exposed to cycles of hyperthermia for 90 min followed by a rest period of 15 min. During this period mice have easy access to food and water. High body temperature spikes in the mice during first few hyperthermia exposure cycles are prevented by immobilizing the animal. Additionally, normal saline is administered in first few cycles to minimize the effects of dehydration. This protocol can simulate fever like conditions in mice up to 12-24 hr. We have used 8-12 weeks old BALB/Cj female mice to demonstrate the protocol.

This paper describes a protocol for whole body hyperthermia in mice that can stimulate fever like conditions up to 12-24 hr.

Protokoll für Long Duration Ganzkörper-Hyperthermie in Mäuse

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body ...

Use of Animal Model of Sepsis to Evaluate Novel Herbal Therapies

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several techniques, including infusion of exogenous bacterial toxin (endotoxemia) or bacteria (bacteremia), as well as surgical perforation of the cecum by cecal ligation and puncture (CLP)1-3. CLP allows bacteria spillage and fecal contamination of the peritoneal cavity, mimicking the human clinical disease of perforated appendicitis or diverticulitis. The severity of sepsis, as reflected by the eventual mortality rates, can be controlled surgically by varying the size of the needle used for cecal puncture2. In animals, CLP induces similar, biphasic hemodynamic cardiovascular, metabolic, and immunological responses as observed during the clinical course of human sepsis3. Thus, the CLP model is considered as one of the most clinically relevant models for experimental sepsis1-3.
Various animal models have been used to elucidate the intricate mechanisms underlying the pathogenesis of experimental sepsis. The lethal consequence of sepsis is attributable partly to an excessive accumulation of early cytokines (such as TNF, IL-1 and IFN-&gamma;)4-6 and late proinflammatory mediators (e.g., HMGB1)7. Compared with early proinflammatory cytokines, late-acting mediators have a wider therapeutic window for clinical applications. For instance, delayed administration of HMGB1-neutralizing antibodies beginning 24 hours after CLP, still rescued mice from lethality8,9, establishing HMGB1 as a late mediator of lethal sepsis. The discovery of HMGB1 as a late-acting mediator has initiated a new field of investigation for the development of sepsis therapies using Traditional Chinese Herbal Medicine. In this paper, we describe a procedure of CLP-induced sepsis, and its usage in screening herbal medicine for HMGB1-targeting therapies.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection, and can be simulated by a surgical technique termed cecal ligation and puncture (CLP). Here we describe a method to use CLP-induced animal model to screen medicinal herbs for therapeutic agents.

Die Verwendung Tiermodell für Sepsis zu neuartigen Therapien zu beurteilen Herbal

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several ...

Visualization and Analysis of Blood Flow and Oxygen Consumption in Hepatic Microcirculation: Application to an Acute Hepatitis Model

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of the hepatic blood supply is poorly oxygenated portal vein blood derived from the gastrointestinal tract and spleen. Oxygen is delivered to hepatocytes by blood flowing from a terminal branch of the portal vein to a central venule via sinusoids, and this makes an oxygen gradient in hepatic lobules. The oxygen gradient is an important physical parameter that involves the expression of enzymes upstream and downstream in hepatic microcirculation, but the lack of techniques for measuring oxygen consumption in the hepatic microcirculation has delayed the elucidation of mechanisms relating to oxygen metabolism in liver. We therefore used FITC-labeled erythrocytes to visualize the hepatic microcirculation and used laser-assisted phosphorimetry to measure the partial pressure of oxygen in the microvessels there. Noncontact and continuous optical measurement can quantify blood flow velocities, vessel diameters, and oxygen gradients related to oxygen consumption in the liver. In an acute hepatitis model we made by administering acetaminophen to mice we observed increased oxygen pressure in both portal and central venules but a decreased oxygen gradient in the sinusoids, indicating that hepatocyte necrosis in the pericentral zone could shift the oxygen pressure up and affect enzyme expression in the periportal zone. In conclusion, our optical methods for measuring hepatic hemodynamics and oxygen consumption can reveal mechanisms related to hepatic disease.

An optical system was developed to visualize hepatic microcirculation with FITC-labeled erythrocytes and to measure the partial pressure of oxygen in the microvessels with laser-assisted phosphorimetry. This method can be used to investigate physiological and pathological mechanisms by analyzing microvascular structure, diameter, blood flow velocity, and oxygen tension.

Visualisierung und Analyse des Blutflusses und der Sauerstoffverbrauch in hepatischen Mikrozirkulation: Anwendung auf eine akute Hepatitis-Modell

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of ...

Implanting Glass Spinal Cord Windows in Adult Mice with Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) in adult rodents is the standard experimental model for studying autonomic demyelinating diseases such as multiple sclerosis. Here we present a low-cost and reproducible glass window implantation protocol that is suitable for intravital microscopy and studying the dynamics of spinal cord cytoarchitecture with subcellular resolution in live adult mice with EAE. Briefly, we surgically expose the vertebrae T12-L2 and construct a chamber around the exposed vertebrae using a combination of cyanoacrylate and dental cement. A laminectomy is performed from T13 to L1, and a thin layer of transparent silicone elastomer is applied to the dorsal surface of the exposed spinal cord. A modified glass cover slip is implanted over the exposed spinal cord taking care that the glass does not directly contact the spinal cord. To reduce the infiltration of inflammatory cells between the window and spinal cord, anti-inflammatory treatment is administered every 2 days (as recommended by ethics committee) for the first 10 days after implantation. EAE is induced only 2-3 weeks after the cessation of anti-inflammatory treatment.
Using this approach we successfully induced EAE in 87% of animals with implanted windows and, using Thy1-CFP-23 mice (blue axons in dorsal spinal cord), quantified axonal loss throughout EAE progression. Taken together, this protocol may be useful for studying the recruitment of various cell populations as well as their interaction dynamics, with subcellular resolution and for extended periods of time. This intravital imaging modality represents a valuable tool for developing therapeutic strategies to treat autoimmune demyelinating diseases such as EAE.

We describe a method for implanting and maintaining glass windows over the exposed spinal cords of adult mice for studying experimental autoimmune encephalomyelitis. These windows provide chronic optical access to the spinal cord for monitoring cell dynamics at the subcellular level in live animals using two-photon microscopy.

Experimental autoimmune encephalomyelitis (EAE) in adult rodents is the standard experimental model for studying autonomic demyelinating diseases such as ...

A Magnetic Microbead Occlusion Model to Induce Ocular Hypertension-Dependent Glaucoma in Mice

The use of rodent models of glaucoma has been essential to understand the molecular mechanisms that underlie the pathophysiology of this multifactorial neurodegenerative disease. With the advent of numerous transgenic mouse lines, there is increasing interest in inducible murine models of ocular hypertension. Here, we present an occlusion model of glaucoma based on the injection of magnetic microbeads into the anterior chamber of the eye using a modified microneedle with a facetted bevel. The magnetic microbeads are attracted to the iridocorneal angle using a handheld magnet to block the drainage of aqueous humour from the anterior chamber. This disruption in aqueous dynamics results in a steady elevation of intraocular pressure, which subsequently leads to the loss of retinal ganglion cells, as observed in human glaucoma patients. The microbead occlusion model presented in this manuscript is simple compared to other inducible models of glaucoma and also highly effective and reproducible. Importantly, the modifications presented here minimize common issues that often arise in occlusion models. First, the use of a bevelled glass microneedle prevents backflow of microbeads and ensures that minimal damage occurs to the cornea during the injection, thus reducing injury-related effects. Second, the use of magnetic microbeads ensures the ability to attract most beads to the iridocorneal angle, effectively reducing the number of beads floating in the anterior chamber avoiding contact with other structures (e.g., iris, lens). Lastly, the use of a handheld magnet allows flexibility when handling the small mouse eye to efficiently direct the magnetic microbeads and ensure that there is little reflux of the microbeads from the eye when the microneedle is withdrawn. In summary, the microbead occlusion mouse model presented here is a powerful investigative tool to study neurodegenerative changes that occur during the onset and progression of glaucoma.

Here, we present a protocol to induce ocular hypertension in the murine eye that results in the loss of retinal ganglion cells as observed in glaucoma. Magnetic microbeads are injected into the anterior chamber and attracted to the iridocorneal angle using a magnet to block the outflow of aqueous humour.

Ein Magnet Microbead Occlusion Modell Ocular Hypertension Abhängige Glaucoma in Mäusen zu induzieren

The use of rodent models of glaucoma has been essential to understand the molecular mechanisms that underlie the pathophysiology of this multifactorial ...

Balloon-based Injury to Induce Myointimal Hyperplasia in the Mouse Abdominal Aorta

The use of animal models is essential for a better understanding of MH, one major cause for arterial stenosis.In this article, we demonstrate a murine balloon denudation model, which is comparable with established vessel injury models in large animals. The aorta denudation model with balloon catheters mimics the clinical setting and leads to comparable pathobiological and physiological changes. Briefly, after performing a horizontal incision in the aorta abdominalis, a balloon catheter will be inserted into the vessel, inflated, and introduced retrogradely. Inflation of the balloon will lead to intima injury and overdistension of the vessel. After removing the catheter, the aortic incision will be closed with single stiches. The model shown in this article is reproducible, easy to perform, and can be established quickly and reliably. It is especially suitable for evaluating expensive experimental therapeutic agents, which can be applied in an economical fashion. By using different knockout-mouse strains, the impact of different genes on MH development can be assessed.

This article demonstrates a murine model to study the development of myointimal hyperplasia (MH) after aortic balloon injury.

Ballon-basierte Verletzungen induzieren Myointimal Hyperplasie in der Bauchschlagader Maus

The use of animal models is essential for a better understanding of MH, one major cause for arterial stenosis.In this article, we demonstrate a murine ...

A Closed-chest Model to Induce Transverse Aortic Constriction in Mice

Research on cardiac hypertrophy and heart failure is frequently based on pressure overload mouse models induced by TAC. The standard procedure is to perform a partial thoracotomy to visualize the transverse aortic arch. However, the surgical trauma caused by the thoracotomy in open-chest models changes the respiratory physiology as the ribs are dissected and left unattached after chest closure. To prevent this, we established a minimally invasive, closed chest approach via lateral thoracotomy. Herein we approach the aortic arch via the 2nd intercostal space without entering the chest cavities, leaving the mouse with a less traumatic injury to recover from. We perform this operation using standard laboratory settings for open chest TAC procedures with equal survival rates. Apart from maintaining physiological breathing patterns due to the closed chest approach, the mice seem to benefit by showing rapid recovery, as the less invasive technique appears to facilitate a fast healing process and to reduce immune response after trauma.

Here, we present a protocol of Transverse Aortic constriction (TAC) via a lateral thoracotomy. This technique is a minimally invasive, closed chest surgical procedure aiming to simulate pressure overload and heart failure in mice utilizing standard TAC laboratory settings.

Eine geschlossen Kasten Modell quer Aortenstenose Verengung bei Mäusen zu induzieren

Research on cardiac hypertrophy and heart failure is frequently based on pressure overload mouse models induced by TAC. The standard procedure is to ...

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 ...