Questo lavoro è supportato dall&#39;Università Goethe Hospital Francoforte e una sovvenzione della Fondazione tedesca per la ricerca di 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>

Gli autori non hanno nulla da rivelare.

In studi precedenti, l&#39;epatite sperimentale è stato spesso segnalato per essere solo transitori e molti modelli attuali di una patologia epatica autoimmune dipende da una malattia piuttosto complessi protocolli di induzione (per una rassegna si veda 3,5). Ad esempio, parecchi modelli utilizzare topi transgenici esprimendo antigeni bersaglio specifici e adoptively trasferiti bersaglio antigene-specifici, per lo più TcR-transgenici, cellule T 14,15. Spesso un&#39;infezione supplementare con virus livertropic, batteri o parassiti è necessario di indurre la malattia 16-18. In alternativa, DNA-vaccinazione con plasmidi codificanti per antigeni bersaglio, compresi CYP2D6 19, viene usato per indurre epatite. Tuttavia, una vaccinazione supplementare con plasmidi codificano per pro-infiammatorie citochine, come IL-12, le è richiesto 20. Purtroppo, con poche eccezioni 15,20 epatite è solo transitoria. Il modello di CYP2D6 6,21 utilizza un metodo semplice tO indurre epatite autoimmune semplicemente infettando i topi con un adenovirus esprimere hCYP2D6, l&#39;autoantigene importante EAI-2 7,8. Consegna di CYP2D6 da uno costrutto adenovirus garantisce sia un targeting diretto del fegato nonché una infiammazione locale che promuove la ripartizione di tolleranza. È importante notare tuttavia che sia il titolo virus così come la via di somministrazione è fondamentale per questo modello. Da un lato, Ad-2D6 è un virus replicazione deficiente, così, un titolo sufficiente elevato di virus è richiesto per generare una quantità critica di antigene all&#39;interno del fegato. D&#39;altra parte, troppo alto un titolo potrebbe comportare una fatale insufficienza epatica acuta. Inoltre, è stato dimostrato precedentemente che infezione di topi da parte di alti titoli di adenovirus porta ad un esaurimento funzionale della risposta immunitaria 22. Nel nostro modello, abbiamo usato una combinazione di infezione endovenosa e intraperitoneale al fine di ottenere sia infiltrazione cronica cellulare nonché extfibrosi ensive. Abbiamo osservato che l&#39;infezione endovenosa sola provoca ancora massiccia infiltrazione cellulare, ma nessuna fibrosi della regione sottocapsulare, indicando che infiammazione peritoneale a causa dell&#39;iniezione intraperitoneale potessero essere coinvolti in il continuo accumulo sottocapsulare di collagene (Hintermann &amp; Christen, manoscritto in preparazione). Tuttavia, è importante notare che la fibrosi epatica osservata è antigene-specifica, poiché non abbiamo fatto rilevare attivazione delle cellule stellate epatiche e della deposizione collagene dopo infezione con pari virus dei titoli-di Ad-GFP o Ad-Control 23. Il CYP2D6 modello riflette molti aspetti della umana 1,2 AIH, come ad esempio l&#39;epatite cronica, caratterizzata da persistente infiltrazione cellulare e fibrosi epatica 6. Inoltre, la presenza di high-titoli anticorpali anti-CYP2D6 anticorpi con specificità di epitopo simili e diffondendo 13 a LKM-1 anticorpi trovati in AIH-2 pazienti indica la preza di una comune reattività cronica autoimmune. CYP2D6 è il principale antigene in AIH-2, ma esso non viene riconosciuto da pazienti diagnosticati con il più frequente tipo 1 AIH. Inoltre, i pazienti affetti da altre malattie del fegato ad eziologia autoimmune, come la cirrosi biliare primaria (PBC) o colangite sclerosante primaria (PSC), di generare caratteristici autoanticorpi con specificità per autoantigeni fegato distinte e il loro fegato mostrano differenti caratteristiche patologiche di centraggio sulle piccole (PBC ) o grande (PSC) dotti biliari. Tuttavia, i modelli CYP2D6 offre l&#39;opportunità di analizzare i meccanismi coinvolti nei processi di immunopathogenic epatiche infiammatorie croniche che si manifestano nel corso delle malattie autoimmuni del fegato, per identificare i giocatori chiave che guidano la distruzione autoimmune di epatociti e valutare possibili interventi terapeutici per curare la malattia.

<table border="1"><tbody><tr><td> Nome dell&#39;apparecchiatura </td><td> Azienda </td><td> Numero di catalogo </td><td> Comments </td></tr><tr><td> Ago 23G </td><td> BD Biosciences </td><td> 300800 </td><td></td></tr><tr><td> 27 ½ G ago </td><td> VWR </td><td> 612-0151 </td><td></td></tr><tr><td> 30 ago ½ G </td><td> BD Biosciences </td><td> 304000 </td><td></td></tr><tr><td> Strisce reattive alanina aminotransferasi (GPT / ALT) </td><td> Roche Diagnostics </td><td> 10 745 138 202 </td><td></td></tr><tr><td> Strisce reattive aspartato aminotransferasi (GOT / AST) </td><td> Roche Diagnostics </td><td> 10 745 120 202 </td><td></td></tr><tr><td> Anestesia Unità Univentor 400 </td><td> <a href="http://www.agnthos.se" target="_blank" >AgnTho di</a> </td><td></td><td></td></tr><tr><td> Stampi di base, 37x24x10 mm e getta </td><td> VWR </td><td> 720-0208 </td><td></td></tr><tr><td> Cella filtro 70 millimetri </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> Eparinizzati tubi capillari </td><td> Pescatore </td><td> 3123987 </td><td></td></tr><tr><td> Vetrini SuperFrost Plus </td><td> Menzel Gläser </td><td> J1800AMNZ </td><td></td></tr><tr><td> Microtainer SST tubi </td><td> BD Biosciences </td><td> 365951 </td><td></td></tr><tr><td> Micropiastre, V-fondo </td><td> VWR </td><td> 391-1924 </td><td></td></tr><tr><td> Reflotron Plus. </td><td> Roche Diagnostics </td><td></td><td> Determiniation di siero AST / ALT </td></tr><tr><td colspan="4"></td></tr><tr><td> Nome of il reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Comments </td></tr><tr><td> Coniglio anti-topo anti-Collagene di tipo I IgG, </td><td> Chemicon </td><td> AB765P </td><td></td></tr><tr><td> Biotinilato di capra anti-IgG di coniglio </td><td> Vector (AXXORA) </td><td> VC-BA-1000-MC15 </td><td></td></tr><tr><td> FITC-coniugato anticorpo anti topo CD8a </td><td> Southern Biotech </td><td> 1550-1502 </td><td></td></tr><tr><td> PE-coniugato anti topo IFNy </td><td> BD Biosciences </td><td> 554412 </td><td></td></tr><tr><td> PE-coniugato anti-topo CD16/CD32 anticorpo (FcR blocco) </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 Blocco kit </td><td&gt; Vettore (AXXORA) </td><td> VC-SP-2001-KI01 </td><td></td></tr><tr><td> A brefeldina </td><td> Sigma </td><td> B6542 </td><td></td></tr><tr><td> Collagenasi IV </td><td> Sigma </td><td> C5138-100mg </td><td></td></tr><tr><td> DAB substrato kit 3&#39;3&#39;diaminobenzidine </td><td> Vector (AXXORA) </td><td> VC-SK-4100-KI01 </td><td></td></tr><tr><td> DNasi </td><td> Sigma </td><td> DN-25 </td><td></td></tr><tr><td> Elite ABC reagente </td><td> Vector (AXXORA) </td><td> VC-PK-7100-L050 </td><td></td></tr><tr><td> Eosina G / G soluzione </td><td> Roth </td><td> X883.1 </td><td></td></tr><tr><td> Siero fetale bovino (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> Meyer Ematossilina soluzione </td><td> Industry </td><td> A4840, 1000 </td><td></td></tr><tr><td> Ottobre Compound </td><td> Sakura Finetek Europa </td><td> 4583 </td><td></td></tr><tr><td> PBS-tamponata formaldeide 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> 6.638,1 </td><td></td></tr><tr><td> RPMI </td><td> Invitrogen </td><td> 61870 </td><td></td></tr><tr><td> Saponina dalla corteccia Quillaja </td><td> Sigma </td><td> S7900 </td><td></td></tr></tbody></table>

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

L&#39;epatite autoimmune è una malattia rara ma pericolosa per la vita autoimmune del fegato di 1,2 eziologia sconosciuta. In passato, i tentativi numerosi sono stati fatti per generare un modello animale che riflette le caratteristiche della malattia umana 3-5. Tuttavia, in vari modelli la induzione di malattia era piuttosto complessa e spesso epatite era solo transitoria 3-5. Pertanto, abbiamo sviluppato un modello semplice topo che utilizza l&#39;autoantigene maggiore umano in tipo 2 epatite autoimmune (AIH-2), cioè hCYP2D6, come un trigger 6. Di tipo 1 di fegato e rene anticorpi microsomiali (LKM-1) anticorpi che riconoscono hCYP2D6 sono il segno distintivo di EAI-2 7,8. Consegna di tipo selvatico hCYP2D6 in FVB o C57BL / 6 topi era da un costrutto Adenovirus (Ad-2D6) che assicura una consegna diretta dell&#39;antigene di attivazione al fegato. Così, l&#39;infiammazione conseguente locale genera un campo fertile 9 per il successivo sviluppo di autoimmunità. A combination di iniezione endovenosa e intraperitoneale di Ad-2D6 è la via più efficace per indurre un danno a lungo termine autoimmune al fegato (sezione 1). Qui forniamo un protocollo dettagliato su come autoimmune malattia epatica viene indotta nel modello CYP2D6 e come i differenti aspetti di danno epatico può essere valutata. In primo luogo, i livelli sierici di markers che indicano distruzione epatociti, come aminotransferasi, nonché i titoli di hCYP2D6 anticorpi sono determinate campionando sangue retroorbitaly (sezione 2). In secondo luogo, il hCYP2D6-specifica risposta di cellule T è caratterizzata da raccogliendo linfociti dalla milza e il fegato. Al fine di ottenere linfociti fegato puri, i fegatini sono perfusi dalla PBS attraverso la vena portale (sezione 3), digerito in collagene e purificato su un gradiente di Percoll (sezione 4). La frequenza di hCYP2D6 specifiche cellule T viene analizzato dalla stimolazione con hCYP2D6 peptidi e l&#39;identificazione delle cellule che producono IFN-mediante citometria di flusso (sezione 5). Terzo,infiltrazione cellulare e fibrosi è determinata dalla immunoistochimica su sezioni di fegato (sezione 6). Regime Tale analisi dovrà essere condotta in diversi momenti dopo l&#39;inizio della malattia al fine di dimostrare la natura cronica del modello. La grandezza della risposta immunitaria caratterizzata dalla frequenza e l&#39;attività di hCYP2D6-specifici T e / o cellule B e il grado del danno epatico e fibrosi devono essere valutati per una successiva valutazione di possibili trattamenti per prevenire, ritardare o abrogare la autodestructive processo del fegato.

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

Infezione di topi con uno Adenovirus che esprime il autoantigene maggiore umano citocromo P450 2D6 (hCYP2D6) riconosciuti da sieri di pazienti affetti da di tipo 2 risultati epatite autoimmune in una forma persistente di autoimmune-mediata malattia epatica caratterizzata da estensiva epatite, fibrosi e la generazione di uno CYP2D6 -specifica risposta immunitaria.

Il CYP2D6 modello animale: come indurre epatite autoimmune in topi

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 ...

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

Research

JoVE Journal

Medicine

18.9K Views.  Goethe University Hospital Frankfurt. Infezione di topi con uno Adenovirus che esprime il autoantigene maggiore umano citocromo P450 2D6 (hCYP2D6) riconosciuti da sieri di pazienti affetti da di tipo 2 risultati epatite autoimmune in una forma persistente di autoimmune-mediata malattia epatica caratterizzata da estensiva epatite, fibrosi e la generazione di uno CYP2D6 -specifica risposta immunitaria.

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.

Pressione di ventilazione controllata per indurre lesioni polmonari acute nei topi

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

Ricerca

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.

Biomarcatori in un modello animale per Rivelare correlati neurali, ematologiche, e comportamentali di 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.

Protocollo per lungo Ipertermia Corpo intera durata in Mouse

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.

Uso di un modello animale di sepsi per valutare nuove terapie a base di erbe

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.

Visualizzazione e analisi del flusso ematico e consumo di ossigeno nella microcircolazione epatica: Applicazione di un modello di epatite acuta

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.

Un magnetica microperla occlusione modello per indurre ipertensione oculare-Dependent Glaucoma nei topi

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.

Basato su palloncino ferita per indurre iperplasia Myointimal nell'Aorta addominale di Mouse

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 modello chiuso-cassa per indurre costrizione trasversale aortica in topi

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.

Misurazione di contrattilità isometrica dell'arteria mesenterica Mouse con filo Myography

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