Este trabajo es apoyado por el Goethe Hospital Universitario de Frankfurt y una beca de la Fundación Alemana de Investigación de la Universidad de California

<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>

Los autores no tienen nada que revelar.

En estudios anteriores, la hepatitis experimental, se informó a menudo para ser sólo los actuales modelos transitorios y muchos de la enfermedad autoinmune del hígado depende de un protocolo de inducción más complejos de enfermedades (para revisiones, véase 3,5). Por ejemplo, varios modelos de uso de ratones transgénicos que expresan antígenos específicos de destino y adoptively transferido objetivo de antígeno-específicos, en su mayoría TCR transgénicos, las células T 14,15. A menudo una infección adicional con el virus de livertropic, bacterias o parásitos es necesario para inducir la enfermedad 16-18. Alternativamente, el ADN-la vacunación con plásmidos que codifican para antígenos diana, incluyendo CYP2D6 19, se utiliza para inducir la hepatitis. Sin embargo, una vacunación adicional con plásmidos que codifican para citoquinas pro-inflamatorias, como la IL-12, se requiere 20. Por desgracia, con pocas excepciones 15,20 hepatitis es sólo transitoria. El modelo del CYP2D6 6,21 utiliza un t método sencilloo inducir a la hepatitis autoinmune, simplemente infectar a los ratones con un adenovirus que expresa hCYP2D6, el autoantígeno mayor en la HAI-2 7,8. Entrega de CYP2D6 por un constructo de adenovirus garantiza tanto una focalización directa del hígado así como una inflamación local que promueve el desglose de la tolerancia. Es importante señalar sin embargo que tanto el título del virus, así como la vía de administración es crucial para este modelo. Por un lado, Ad-2D6 es un virus de replicación deficiente, por lo tanto, un título suficiente alta de virus se requiere para generar una cantidad crítica de antígeno dentro del hígado. Por otro lado, demasiado alto un título podría resultar en una insuficiencia hepática aguda fatal. Además, se ha demostrado previamente que la infección de ratones por títulos altos de adenovirus conduce a un agotamiento funcional de la respuesta inmune 22. En nuestro modelo, hemos utilizado una combinación de infección por vía intravenosa e intraperitoneal con el fin de obtener tanto la infiltración crónica celular, así como extLa fibrosis ensive. Hemos observado que la infección por vía intravenosa sola todavía provoca la infiltración celular masiva, pero no la fibrosis de la región subcapsular, lo que indica que la inflamación peritoneal, debido a la inyección intraperitoneal podrían estar involucrados en la continua acumulación subcapsular de colágeno (Hintermann y Christen, manuscrito en preparación). Sin embargo, es importante señalar que la fibrosis hepática observada es antígeno-específica, ya que no detectó la activación de las células estrelladas hepáticas y la deposición de colágeno después de la infección con la igualdad de virus títulos de Ad-GFP o Ad-Control de 23. El modelo de CYP2D6 refleja muchos aspectos de la humana 1,2 HAI, como la hepatitis crónica caracterizada por la infiltración celular y la persistencia de la fibrosis hepática 6. Además, la presencia de títulos elevados de anti-CYP2D6 anticuerpos con especificidad epítopo similar y extendiendo 13 a LKM-1 anticuerpos encontrados en la HAI-2 pacientes indica la presencia de un sistema común de reactividad autoinmune crónica. CYP2D6 es el principal antígeno en la HAI-2, pero no es reconocida por pacientes diagnosticados con el tipo más frecuente de una hepatitis autoinmune. Además, los pacientes que sufren de otras enfermedades del hígado de etiología autoinmune, como la cirrosis biliar primaria (CBP) o colangitis esclerosante primaria (PSC), generación de autoanticuerpos sello con la especificidad de distintos autoantígenos del hígado y el hígado muestran diferentes características patológicas de centrado en las pequeñas (PBC ) o grande (PSC), los conductos biliares. Sin embargo, los modelos de CYP2D6 ofrece la oportunidad de analizar mecanismos inmunopatológicos implicados en los procesos inflamatorios crónicos hepáticos, como ocurre en las enfermedades autoinmunes del hígado, para identificar a los actores clave que impulsan la destrucción autoinmune de los hepatocitos y evaluar posibles intervenciones terapéuticas para curar la enfermedad.

<table border="1"><tbody><tr><td> Nombre del equipo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> Aguja de 23G </td><td> BD Biosciences </td><td> 300800 </td><td></td></tr><tr><td> 27 ½ G aguja </td><td> VWR </td><td> 612-0151 </td><td></td></tr><tr><td> 30 ½ aguja de calibre </td><td> BD Biosciences </td><td> 304000 </td><td></td></tr><tr><td> Alanina aminotransferasa tiras reactivas (GPT / ALT) </td><td> Roche Diagnostics </td><td> 10 745 138 202 </td><td></td></tr><tr><td> Aspartato aminotransferasa tiras reactivas (GOT / AST) </td><td> Roche Diagnostics </td><td> 10 745 120 202 </td><td></td></tr><tr><td> Anestesia Unidad Univentor 400 </td><td> <a href="http://www.agnthos.se" target="_blank" >AgnTho de</a> </td><td></td><td></td></tr><tr><td> Moldes de base, 37x24x10 mm desechables </td><td> VWR </td><td> 720-0208 </td><td></td></tr><tr><td> Celular filtro de 70mm </td><td> VWR </td><td> 734-0003 </td><td></td></tr><tr><td> Criostato </td><td> Leica </td><td> CM1850 UV </td><td></td></tr><tr><td> Tubos capilares heparinizados </td><td> Pescador </td><td> 3123987 </td><td></td></tr><tr><td> Portaobjetos Superfrost Plus </td><td> Menzel Gläser </td><td> J1800AMNZ </td><td></td></tr><tr><td> Microtainer SST tubos </td><td> BD Biosciences </td><td> 365951 </td><td></td></tr><tr><td> Placas de microtitulación, fondo en V </td><td> VWR </td><td> 391-1924 </td><td></td></tr><tr><td> Reflotron </td><td> Roche Diagnostics </td><td></td><td> Determiniation de AST / ALT </td></tr><tr><td colspan="4"></td></tr><tr><td> Nombre of el reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> De conejo anti-ratón anti-colágeno tipo I IgG, </td><td> Chemicon </td><td> AB765P </td><td></td></tr><tr><td> Biotinilado de cabra anti-IgG de conejo </td><td> Vector (AXXORA) </td><td> VC-BA-1000-MC15 </td><td></td></tr><tr><td> FITC-conjugado de anticuerpos de ratón anti CD8a </td><td> Southern Biotech </td><td> 1550-1502 </td><td></td></tr><tr><td> PE-ratón conjugado con anticuerpos anti IFN </td><td> BD Biosciences </td><td> 554412 </td><td></td></tr><tr><td> PE-conjugado anti-ratón CD16/CD32 anticuerpos (FcR bloque) </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> Avidina / biotina kit de bloqueo </td><td&gt; Vectores (AXXORA) </td><td> VC-SP-2001-KI01 </td><td></td></tr><tr><td> Un Brefeldin </td><td> Sigma </td><td> B6542 </td><td></td></tr><tr><td> Colagenasa IV </td><td> Sigma </td><td> C5138-100MG </td><td></td></tr><tr><td> DAB sustrato kit 3&#39;3&#39;diaminobenzidine </td><td> Vector (AXXORA) </td><td> VC-SK-4100-KI01 </td><td></td></tr><tr><td> DNasa </td><td> Sigma </td><td> DN-25 </td><td></td></tr><tr><td> Elite ABC reactivo </td><td> Vector (AXXORA) </td><td> VC-PK-7100-L050 </td><td></td></tr><tr><td> Eosina G / Y la solución </td><td> Roth </td><td> X883.1 </td><td></td></tr><tr><td> De suero fetal de ternero (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> Solución de hematoxilina de Meyer </td><td> Applichem </td><td> A4840, 1000 </td><td></td></tr><tr><td> Compuesto octubre </td><td> Sakura Finetek Europa </td><td> 4583 </td><td></td></tr><tr><td> PBS-formaldehído tamponado al 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> La saponina de la corteza de quillay </td><td> Sigma </td><td> S7900 </td><td></td></tr></tbody></table>

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

La hepatitis autoinmune es una enfermedad autoinmune poco frecuente, pero que amenaza la vida del hígado de etiología desconocida 1,2. En los muchos intentos pasados ​​se han hecho para generar un modelo animal que refleja las características de la enfermedad humana 3-5. Sin embargo, en varios modelos de la inducción de la enfermedad era bastante compleja y con frecuencia la hepatitis era sólo transitoria 3-5. Por lo tanto, hemos desarrollado un modelo de ratón sencillo que utiliza el autoantígeno humano importante en tipo 2 hepatitis autoinmune (HAI-2), a saber hCYP2D6, como un gatillo 6. Tipo 1 de hígado-riñón anticuerpos microsomales (LKM-1) anticuerpos que reconocen hCYP2D6 son el sello distintivo de la HAI-2 7,8. La entrega de hCYP2D6 en wildtype FVB o ratones C57BL / 6 fue por una construcción de adenovirus (Ad-2D6) que asegura una entrega directa del antígeno desencadenante para el hígado. Por lo tanto, la inflamación subsiguiente local genera un campo fértil 9 para el posterior desarrollo de la autoinmunidad. A combination de la inyección por vía intravenosa e intraperitoneal de Ad-2D6 es la vía más eficaz para inducir un daño autoinmune de larga duración en el hígado (sección 1). A continuación presentamos un protocolo detallado de cómo la enfermedad hepática autoinmune es inducida en el modelo de CYP2D6 y cómo los diferentes aspectos de daño hepático puede ser evaluada. En primer lugar, los niveles séricos de marcadores que indican destrucción de hepatocitos, tales como las aminotransferasas, así como los títulos de hCYP2D6 anticuerpos se determinó mediante el muestreo de sangre retroorbitaly (sección 2). En segundo lugar, la respuesta hCYP2D6-específica de células T se caracteriza por recoger los linfocitos desde el bazo y el hígado. A fin de obtener linfocitos puros hígado, los hígados se perfundieron por PBS a través de la vena porta (sección 3), digerido en el colágeno y purifica sobre un gradiente de Percoll (sección 4). La frecuencia de las células T específicas de hCYP2D6 es analizada por la estimulación con péptidos hCYP2D6 y la identificación de las células productoras de IFN por citometría de flujo (sección 5). En tercer lugar,infiltración celular y la fibrosis se determina mediante técnicas de inmunohistoquímica de secciones de hígado (sección 6). Régimen de tipo de análisis tiene que ser llevado a cabo en varias ocasiones después de la iniciación de la enfermedad con el fin de probar la naturaleza crónica de la modelo. La magnitud de la respuesta inmune caracterizada por la frecuencia y la actividad de hCYP2D6-específicas T y / o células B y el grado de la lesión hepática y fibrosis tienen que ser evaluados para una posterior evaluación de posibles tratamientos para prevenir, retrasar o abrogar el autodestructiva proceso del hígado.

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

La infección de ratones con un adenovirus que expresan el autoantígeno humano importante del citocromo P450 2D6 (hCYP2D6) reconocidos por los sueros de los pacientes con tipo 2, los resultados de hepatitis autoinmune en una forma persistente de la enfermedad hepática autoinmune mediada por la hepatitis se caracteriza por una amplia fibrosis, y la generación de un CYP2D6 la respuesta inmune específica.

El CYP2D6 Animal Modelo: cómo inducir a la hepatitis autoinmune en ratones

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 vistas.  Goethe University Hospital Frankfurt. La infección de ratones con un adenovirus que expresan el autoantígeno humano importante del citocromo P450 2D6 (hCYP2D6) reconocidos por los sueros de los pacientes con tipo 2, los resultados de hepatitis autoinmune en una forma persistente de la enfermedad hepática autoinmune mediada por la hepatitis se caracteriza por una amplia fibrosis, y la generación de un CYP2D6 la respuesta inmune específica.

Video: 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.

La presión de la ventilación controlada para inducir la lesión pulmonar aguda en ratones

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

Investigación

Medicina

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.

Biomarcadores en un modelo animal para Revelar correlatos neurales, hematológicas y conductuales de estrés postraumático

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.

Protocolo para la hipertermia corporal Larga Duración Total en ratones

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.

El uso de modelos animales de sepsis para evaluar nuevas terapias a base de plantas

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.

Visualización y análisis de flujo de la sangre y del consumo de oxígeno en la microcirculación hepática: Aplicación a un Modelo de cuadro de hepatitis aguda

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.

A Magnetic Microbead Oclusión Modelo para inducir hipertensión ocular dependiente de glaucoma en ratones

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.

Lesión basada en globo para inducir la hiperplasia Myointimal de la Aorta Abdominal de ratón

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.

Un modelo del cerrado-pecho para inducir constricción aórtica transversal en ratones

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.

Medición de la contractilidad isométrica de la arteria mesentérica de ratón con cable miografía

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