Name: murine distal colostomy, a novel model of diversion colitis in c57bl/6 mice
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Tutti i metodi descritti qui sono stati approvati dall'autorità di governo veterinaria (Landesamt für Landwirtschaft, Lebensmittelsicherheit und Fischerei Mecklenburg-Vorpommern, (LALLF M-V)).
1. cura preoperatoria e la preparazione dell'animale
<ol><li>All'arrivo nella struttura degli animali, animali (C57Bl/6) si divide in gruppi di dimensioni simili, ogni gruppo della gabbia insieme e mantenere gruppi costante durante gli esperimenti. Nota: Utilizzare animali dello stesso sesso. I risultati descritti sono stati ottenuti con topi maschi. Nota: Se vengono utilizzati gli animali maschii, gruppi gabbia insieme a partire da quando sono 7 settimane di età in modo che possa essere stabilita una gerarchia, minimizzando così il rischio di comportamento aggressivo durante gli esperimenti.</li>
	<li>Almeno una settimana prima dell'intervento chirurgico, passare a un'ad alta energia (> 14 MJ/kg) e ad alta percentuale proteica (> 20%) alimentazione contenente tutti gli oligoelementi essenziali e vitamine (per informazioni dettagliate, vedere elenco dei materiali). Nota: Assicurare tutti i topi pesare almeno 25 g quando l'ambulatorio è effettuato.</li>
	<li>Indurre l'anestesia e analgesia tramite l'iniezione intraperitoneale di ketamina (87 mg/kg i.p.) e xilazina cloridrato (13 mg/kg i.p.). Attendere fino a quando il mouse tollera lo stimolo meccanico, per esempio punta passo, senza risposta motoria.</li>
	<li>Fissare il mouse narcotizzato con nastri in posizione supina su un sottofondo di calore posizionato presso il banco di funzionamento, garantendo stabile posizionamento durante il funzionamento ed evitare un'enorme perdita di calore corporeo. Nota: Il sottoposto di calore deve avere una temperatura di 36 ° C a 40 ° C; la temperatura ambiente di funzionamento dovrebbe essere 21 ° C.</li>
</ol>2. operazione distale colostomia
<ol><li>Radersi i capelli addominali. Prima di iniziare l'intervento chirurgico, disinfettare il campo di funzionamento tre volte utilizzando alcol 70% e un iodophor. Drappo il campo di funzionamento per garantire condizioni di asepsi.</li>
	<li>Per eseguire una laparotomia mediana di 15 mm, incidendo i muscoli addominali ed il peritoneum lungo la linea alba, riducendo così al minimo la perdita di sangue.</li>
	<li>Utilizzare due DeBakey atraumatica, estrarre con cautela l'intestino cieco, ileo terminale e ascendente e colon trasverso dalla cavità peritoneale. Nota: Fare attenzione a limitare rigorosamente la manipolazione meccanica dell'intestino per evitare lesioni alle strutture mesenteriche.</li>
	<li>Identificare il Polo cecal, il colon ascendente e piccolo intestino (figure 1a e 1b). Nota: La corretta identificazione del colon ascendente è fondamentale per il corretto posizionamento del colostomy. Nei casi in cui l'anatomia della regione ileocecal è ambigua, la presenza di placche di Peyer identifica l'intestino tenue e la presenza di feci formate caratterizza il colon.</li>
	<li>Usare un righello per determinare la posizione del colostomy futuri. Esso va posizionata a 20 mm distale alla valvola ileocecal per una colostomia distale.</li>
	<li>Praticare una seconda incisione di 3 mm nella parete addominale nel quadrante superiore destro. Tirare il segmento del colon precedentemente identificato attraverso questa incisione per formare un anello, facendo attenzione a non distorcere il ciclo.</li>
	<li>Passare accuratamente una cannula 22 calibri flessibili i.v. attraverso il mesocolon. Fare attenzione a non per danneggiare le strutture vascolari mesenteriche.</li>
	<li>Restituire l'intestino nella cavità peritoneale.</li>
	<li>Difficoltà entrambe le estremità del tubo flessibile alla cute tramite semplici punti di sutura e una sutura riassorbibile (ad es., polyglactin 910 o polyfil 4-0 1/2 c).</li>
	<li>Prima di chiudere la laparotomia, eseguire la rianimazione fluida usando un'iniezione intraperitoneale di 0,5 mL di soluzione fisiologica 0,9%.</li>
	<li>Chiudere il peritoneo e lo strato del muscolo con una sutura continua utilizzando una sutura riassorbibile (ad es., polyglactin 910 o polyfil 4-0 1/2 c). Chiudere la pelle con una sutura continua utilizzando una sutura riassorbibile (ad es., polyglactin 910 o polyfil 4-0 1 / 2c).</li>
	<li>Aprire il ciclo colon esteriorizzato eseguendo un transection subtotale utilizzando una forbice bene. Evitare tutte le lesioni al mesentery. Transetto non colon completamente.</li>
	<li>Difficoltà ogni apertura di colostomia con tre punti di pieno-spessore singoli al peritoneo e della pelle utilizzando un monofilo, suturare assorbibile (ad es., polidioxanone o monofilo 6-0 3/8s). Ciclo afferente, che è un colostomy dell'estremità-funzionale, e il ciclo efferente, che è una fistola mucosa, sono chiaramente separati a questo punto (Figura 1C). Nota: Tempo di funzionamento deve essere meno di 20 minuti per limitare le perdite di fluido e termiche.</li>
	<li>Dopo chirurgia, di finitura disinfettare strumenti utilizzando una soluzione di disinfezione senza aldeide in bagno ad ultrasuoni secondo le istruzioni del produttore.</li>
</ol>3. sham operazione (Colotomy)
<ol><li>Eseguire i passaggi 1.1. attraverso 2.4.</li>
	<li>Usare un righello per determinare la posizione di futuri colotomy. Il colotomy deve essere posizionato alla stessa distanza dalla valvola ileocecal come la colostomia nel gruppo sperimentale.</li>
	<li>Aprire i due punti almeno due terzi utilizzando forbici bene la sua circonferenza.</li>
	<li>Nelle vicinanze il colotomy con un singolo strato, pieno-spessore interrotta sutura utilizzando un monofilo, suturare assorbibile (ad es., polidioxanone, monofilo 6-0 3/8s). Nota: Tempo di funzionamento deve essere meno di 20 minuti per un chirurgo sperimentato, limitando le perdite di fluido e termiche.</li>
	<li>Eseguire i passaggi 2.10. , 2.11. e 2.14.</li>
</ol>4. postoperatoria
<ol><li>Restituire gli animali nelle loro gabbie. Offrono un'atmosfera ben temperata di 37 ° C (ad es., con una lampada a raggi infrarossi) fino a quando i topi sono completamente svegli. Quindi, tenere topi in un ambiente di temperatura e umidità regolata (21 ° C, umidità relativa 30% ± 10%). Consentire il libero accesso a cibo e acqua potabile. Per facilitare l'assorbimento fluido, fornire ulteriori mangimi impregnati d'acqua.</li>
	<li>Iniziare l'analgesia postoperatoria iniettando 0,1 mg/kg corpo peso buprenorfina s.c. quando animali mostrano risposta alla stimolazione meccanica. Fare attenzione a evitare la depressione respiratoria.</li>
	<li>Supplemento acqua potabile con 1 mg/mL tramadol per analgesia continua durante la prima settimana postoperatoria.</li>
	<li>Per compensare la diminuita assunzione di liquidi causa di ridotta mobilità, fornire un solido Pad bere nella gabbia durante la prima settimana postoperatoria.</li>
	<li>Pesare gli animali e comportamento animale punteggio al giorno durante la prima settimana, ogni due giorni durante il resto del primo mese e ogni terzo giorno durante il secondo mese utilizzando il Punteggio di severità di malattia descritto in Kleinwort et al. 6.</li>
</ol>

<ol>
	<li>Glotzer, D. J., Glick, M. E., Goldman, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proctitis+and+colitis+following+diversion+of+the+fecal+stream.">Proctitis and colitis following diversion of the fecal stream.</a> Gastroenterology. 80, 438-441 (1981).</li><li>Morson, B., Dawson, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Gastrointestinal Pathology. , (1972).</li><li>Whelan, R. L., Abramson, D., Kim, D. S., Hashmi, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversion+colitis.+A+prospective+study.">Diversion colitis. A prospective study.</a> Surg Endosc. 8, 19-24 (1994).</li><li>Haas, P. A., Fox, T. A., Szilagy, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoscopic+examination+of+the+colon+and+rectum+distal+to+a+colostomy.">Endoscopic examination of the colon and rectum distal to a colostomy.</a> Am J Gastroenterol. 85, 850-854 (1990).</li><li>Valatas, V., Bamias, G., Kolios, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+colitis+models:+Insights+into+the+pathogenesis+of+inflammatory+bowel+disease+and+translational+issues.">Experimental colitis models: Insights into the pathogenesis of inflammatory bowel disease and translational issues.</a> Eur J Pharmacol. 759, 253-264 (2015).</li><li>Kleinwort, A., D&#246;ring, P., Hackbarth, C., Heidecke, C. D., Schulze, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deviation+of+the+Fecal+Stream+in+Colonic+Bowel+Segments+Results+in+Increased+Numbers+of+Isolated+Lymphoid+Follicles+in+the+Submucosal+Compartment+in+a+Novel+Murine+Model+of+Diversion+Colitis.">Deviation of the Fecal Stream in Colonic Bowel Segments Results in Increased Numbers of Isolated Lymphoid Follicles in the Submucosal Compartment in a Novel Murine Model of Diversion Colitis.</a> Biomed Res Int. 2017, 13 (2017).</li><li>Tangjarukij, C., Navasumrit, P., Zelikoff, J. T., Ruchirawat, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+pyridoxine+deficiency+and+supplementation+on+hematological+profiles,+lymphocyte+function,+and+hepatic+cytochrome+P450+in+B6C3F1+mice.">The effects of pyridoxine deficiency and supplementation on hematological profiles, lymphocyte function, and hepatic cytochrome P450 in B6C3F1 mice.</a> J Immunotoxicol. 6, 147-160 (2009).</li><li>Soave, O., Brand, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coprophagy+in+animals:+a+review.">Coprophagy in animals: a review.</a> Cornell Vet. 81, 357-364 (1991).</li><li>Ebino, K. Y., Yoshinaga, K., Suwa, T., Kuwabara, Y., Takahashi, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+prevention+of+coprophagy+on+pregnant+mice--is+coprophagy+beneficial+on+a+balanced+diet.">Effects of prevention of coprophagy on pregnant mice--is coprophagy beneficial on a balanced diet.</a> Jikken Dobutsu. 38, 245-252 (1989).</li><li>Babickova, J., Tothova, L., Lengyelova, E., Bartonova, A., Hodosy, J., Gardlik, R., Celec, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex+Differences+in+Experimentally+Induced+Colitis+in+Mice:+a+Role+for+Estrogens.">Sex Differences in Experimentally Induced Colitis in Mice: a Role for Estrogens.</a> Inflammation. 38, 1996-2006 (2015).</li><li>Lee, S. M., Kim, N., Son, H. J., Park, J. H., Nam, R. H., Ham, M. H., Choi, D., Sohn, S. H., Shin, E., Hwang, Y. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Effect+of+Sex+on+the+Azoxymethane/Dextran+Sulfate+Sodium-treated+Mice+Model+of+Colon+Cancer.">The Effect of Sex on the Azoxymethane/Dextran Sulfate Sodium-treated Mice Model of Colon Cancer.</a> J Cancer Prev. 21, 271-278 (2016).</li><li>Angele, M. K., Pratschke, S., Hubbard, W. J., Chaudry, I. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gender+differences+in+sepsis:+cardiovascular+and+immunological+aspects.">Gender differences in sepsis: cardiovascular and immunological aspects.</a> Virulence. 5, 12-19 (2013).</li><li>Brown, R. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Social+Behaviour,+reproduction,+and+population+changes+in+the+house+mouse+(Mus+musculus+L).">Social Behaviour, reproduction, and population changes in the house mouse (Mus musculus L).</a> Eccological Monographs. 23, 788-795 (1953).</li><li>Poole, T. B., Morgan, H. D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+in+aggressive+behaviour+betweeen+male+mice+(Mus+musculus+L.)+in+colonies+of+different+sizes.">Differences in aggressive behaviour betweeen male mice (Mus musculus L.) in colonies of different sizes.</a> Animal Behaviour. 21, 788-795 (1973).</li><li>Pasquarelli, N., Voehringer, P., Henke, J., Ferger, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+a+change+in+housing+conditions+on+body+weight,+behavior+and+brain+neurotransmitters+in+male+C57BL/6J+mice.">Effect of a change in housing conditions on body weight, behavior and brain neurotransmitters in male C57BL/6J mice.</a> Behav Brain Res. 333, 35-42 (2017).</li><li>Langgartner, D., Foertsch, S., Fuchsl, A. M., Reber, S. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+and+water+are+not+simple+conditions:+fine+tuning+of+animal+housing+in+male+C57BL/6+mice.">Light and water are not simple conditions: fine tuning of animal housing in male C57BL/6 mice.</a> Stress. 20, 10-18 (2017).</li><li>Nicholson, A., Malcolm, R. D., Russ, P. L., Cough, K., Touma, C., Palme, R., Wiles, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+response+of+C57BL/6J+and+BALB/cJ+mice+to+increased+housing+density.">The response of C57BL/6J and BALB/cJ mice to increased housing density.</a> J Am Assoc Lab Anim Sci. 48, 740-753 (2009).</li><li>Buchler, G., Wos-Oxley, M. L., Smoczek, A., Zschemisch, N. H., Neumann, D., Pieper, D. H., Hedrich, H. J., Bleich, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain-specific+colitis+susceptibility+in+IL10-deficient+mice+depends+on+complex+gut+microbiota-host+interactions.">Strain-specific colitis susceptibility in IL10-deficient mice depends on complex gut microbiota-host interactions.</a> Inflamm Bowel Dis. 18, 943-954 (2012).</li><li>Nakanishi, M., Tazawa, H., Tsuchiya, N., Sugimura, T., Tanaka, T., Nakagama, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+strain+differences+in+inflammatory+responses+of+colonic+mucosa+induced+by+dextran+sulfate+sodium+cause+differential+susceptibility+to+PhIP-induced+large+bowel+carcinogenesis.">Mouse strain differences in inflammatory responses of colonic mucosa induced by dextran sulfate sodium cause differential susceptibility to PhIP-induced large bowel carcinogenesis.</a> Cancer Sci. 98, 1157-1163 (2007).</li><li>Mahler, M., Bristol, I. J., Leiter, E. H., Workman, A. E., Birkenmeier, E. H., Elson, C. O., Sundberg, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+susceptibility+of+inbred+mouse+strains+to+dextran+sulfate+sodium-induced+colitis.">Differential susceptibility of inbred mouse strains to dextran sulfate sodium-induced colitis.</a> Am J Physiol. 274, 544-551 (1998).</li><li>Mekada, K., Abe, K., Murakami, A., Nakamura, S., Nakata, H., Moriwaki, K., Obata, Y., Yoshiki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+differences+among+C57BL/6+substrains.">Genetic differences among C57BL/6 substrains.</a> Exp Anim. 58, 141-149 (2009).</li><li>Sellers, R. S., Clifford, C. B., Treuting, P. M., Brayton, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunological+variation+between+inbred+laboratory+mouse+strains:+points+to+consider+in+phenotyping+genetically+immunomodified+mice.">Immunological variation between inbred laboratory mouse strains: points to consider in phenotyping genetically immunomodified mice.</a> Vet Pathol. 49, 32-43 (2012).</li><li>Mills, C. D., Kincaid, K., Alt, J. M., Heilman, M. J., Hill, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M-1/M-2+macrophages+and+the+Th1/Th2+paradigm.">M-1/M-2 macrophages and the Th1/Th2 paradigm.</a> J Immunol. 164, 6166-6173 (2000).</li></ol>

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Il modello murino di DC ha presentato in questo protocollo affidabile riproduce le caratteristiche istopatologiche del DC umana (ad es., lo sviluppo del de novo di follicoli linfoidi nel submucosa dei segmenti dell'intestino infiammato, cripta accorciamento e riduzioni in numeri delle cellule di calice). Oltre a questo vantaggio, questo modello è indotta da un fattore scatenante molto simile e presenta con un decorso clinico di moderata gravità lieve, come è il caso in esseri umani più colpiti.
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Negli ultimi anni, un numero sostanza di colite non infettive entità diverse da malattie intestinali infiammatorie classica (IBDs; i. e., ulcerosa o colite di Crohn) sono stati caratterizzati clinicamente e histopathologically in esseri umani. I meccanismi fisiopatologici che portano allo sviluppo di queste forme di colite completamente non sono capiti parzialmente perché appropriati modelli animali sono scarse. Colite di diversione è un'entità recentemente descritta. Anche se il termine è stato coniato nel 1980 da Glotzer1, la prima descrizione di un simile fenotipo è stato dato nel 1972 da Morson2. La malattia si sviluppa in 50%-91% dei pazienti con deviazione enterostomia e la sua intensità clinica varia3,4. Data l'incidenza annuale di circa 120.000 pazienti colostomia in Stati Uniti d'America, questa entità di malattia costituisce un problema sanitario importante.
L'obiettivo generale di sviluppare questo protocollo era di fornire un modello murino di DC che si basa su un trigger di colite simile a quello veduto in DC umana e che riproduce le caratteristiche istopatologiche primarie della malattia umana. A differenza di altri modelli di colite murina, induzione di colite nel nostro modello non necessita di animali geneticamente modificati (per esempio, topi transgenici IL-7, N-caderina dominante negativi topi o topi TGFβ- / - ), l'applicazione di chimicamente irritante sostanze (ad es., colite indotta del sodio (DSS) - colite indotta, o acido solfonico del trinitrobenzene (TNBS) - solfato di destrano), o il trasferimento di popolazioni di cellule specifiche in topi carenti immuni (come il trasferimentoad alta CD45RB modello di colite) (per una rassegna, Vedi5). A differenza di altri modelli, il sistema immunitario intatto del nostro modello DC consente la valutazione del meccanismo immunologico coinvolti nello sviluppo del DC. La limitazione della infiammazione delle mucose al segmento di intestino escluso consente la valutazione della sua ripercussione su immunità mucosale in altre parti del tratto gastrointestinale, sull'omeostasi immunitaria in altri scompartimenti immuni del tratto intestinale (per esempio. , Patch di Peyer ed i linfonodi di Intestinum) e sull'omeostasi immunitaria dell'intero organismo. Infine, il nostro modello costituisce uno strumento appropriato per indagare i meccanismi di controllo stimoli infiammatori locali provenienti da cambiamenti nell'ambiente locale normale per entrambi il microbioma locale come pure gli antigeni alimentari.

<table><tbody><tr><td>&lt;em&gt;Operation material&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>heat underlay, 6 watt</td><td>ThermoLux, Witte + Sutor GmbH</td><td>461265</td><td></td></tr><tr><td>Ethanol 70% (with methylethylketon)</td><td>Pharmacie of the University Hospital Greifswald</td><td></td><td></td></tr><tr><td>Wooden applicators, small cotton head onesided, wooden stick, 150 mm length</td><td>Centramed GmbH, Germany</td><td>8308370</td><td>for disinfection of operation field</td></tr><tr><td>Scissor</td><td>Aesculap (BRAUN)</td><td>BC064R</td><td></td></tr><tr><td>Atraumatic DeBakey forceps</td><td>Aesculap (BRAUN)</td><td>OC021R</td><td></td></tr><tr><td>Needle holder</td><td>Aesculap (BRAUN)</td><td>BM012R</td><td></td></tr><tr><td>Vasofix Safety &lt;em&gt;i.v.&lt;/em&gt; cannula 22G</td><td>Braun Melsungen AG, Germany</td><td>4268091S-01</td><td></td></tr><tr><td>VICRYL Plus 4-0 violet braided; V-5 17 m 1/2c; 70 cm</td><td>ETHICON, Inc.</td><td>VCP994H</td><td></td></tr><tr><td>PDS II 6-0 monofil; V-18 13 mm 3/8c; 70 cm</td><td>ETHICON, Inc.</td><td>Z991H</td><td></td></tr><tr><td>BD Plastipak 1 mL (Syringes with needle with sterile interior)</td><td>BD Medical</td><td>305501</td><td></td></tr><tr><td>Triacid-N (N-Dodecylpropan-1,3-diamin)</td><td>ANTISEPTICA, Germany</td><td>18824-01</td><td>disinfection of surgical instruments in ultrasonic bath</td></tr><tr><td>&lt;em&gt;Medications&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>ketamine 10%, 100 mg/mL (ketamine hydrochloride)</td><td>selectavet, Dr. Otto Fischer GmbH</td><td>9089.01.00</td><td>87 mg/kg &lt;em&gt;i.p.&lt;/em&gt;</td></tr><tr><td>Xylasel, 20 mg/mL (xylazine hydrochloride)</td><td>selectavet, Dr. Otto Fischer GmbH</td><td>400300.00.00</td><td>13 mg/kg &lt;em&gt;i.p.&lt;/em&gt;</td></tr><tr><td>NaCl 0.9%</td><td>Braun Melsungen AG, Germany</td><td>6697366.00.00</td><td></td></tr><tr><td>Buprenovet 0.3 mg/mL (buprenorphine)</td><td>Bayer, Germany</td><td>PZN: 01498870</td><td>0.1 mg/kg &lt;em&gt;s.c.&lt;/em&gt;</td></tr><tr><td>Tramal Drops, 100 mg/mL (tramadol hydrochloride)</td><td>Gr&amp;uuml;nenthal GmbH, Germany</td><td>10116838</td><td>1 mg/mL drinking water</td></tr><tr><td>Ceftriaxon-saar 2 g (ceftriaxone)&amp;nbsp;</td><td>Cephasaar GmbH, Germany</td><td>PZN: 08844252</td><td>25 mg/kg body weight &lt;em&gt;i.p.&lt;/em&gt;</td></tr><tr><td>metronidazole 5 mg/mL</td><td>Braun Melsungen AG, Germany</td><td>PZN: 05543515</td><td>12.5 mg/kg body weight &lt;em&gt;i.p.&lt;/em&gt;</td></tr><tr><td>&lt;em&gt;Food&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>ssniff M-Z Ereich</td><td>ssniff, Germany</td><td>&amp;nbsp;V1184-3</td><td></td></tr><tr><td>Solid Drink Dehyprev Vit BIO, pouches</td><td>TripleATrading, the Netherlands</td><td>SDSHPV-75</td><td></td></tr><tr><td>Equipment</td><td></td><td></td><td></td></tr><tr><td>Nikon Eclipse Ci-L</td><td>Nikon Instruments Europe BV, Germany</td><td></td><td>light microscopy</td></tr><tr><td>VetScan HM 5&amp;nbsp;</td><td>Abaxis, USA</td><td>770-9000</td><td>Veterinary hematology analyzer of 50 &amp;micro;l venous EDTA-blood</td></tr><tr><td>Bandelin SONOREX (Ultrasonic bath)</td><td>Bandelin electronics, Germany</td><td>RK 100 H</td><td>disinfection of surgical instruments</td></tr><tr><td>&lt;em&gt;Software&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>NIS-Element BR4 software</td><td>Nikon Instruments Europe BV, Germany</td><td></td><td></td></tr><tr><td>GraphPad Prism Version 6</td><td>GraphPad Software, Inc.</td><td></td><td></td></tr></tbody></table>

murine distal colostomy, a novel model of diversion colitis in c57bl/6 mice

Colite di diversione (DC) è una condizione clinica frequente che si verificano in pazienti con segmenti intestinali esclusi dal flusso a seguito di un'enterostomia diversione fecale. L'eziologia di questa malattia rimane mal definita, ma sembra differire da quello di malattie intestinali infiammatorie classica come il morbo di Crohn e colite ulcerosa. Ricerca finalizzata a decifrare i meccanismi fisiopatologici che portano allo sviluppo di questa malattia è stata gravemente ostacolata dalla mancanza di un appropriato modello murino. Questo protocollo genera un modello murino di DC che facilita lo studio del ruolo del sistema immunitario e la sua interazione con il microbioma nello sviluppo della DC. In questo modello utilizzando animali C57BL/6, parti distali del colon sono esclusi dal flusso fecale creando un colostomy distale, innescare lo sviluppo di delicato per moderare l'infiammazione nei segmenti intestinali esclusi e riproducendo le lesioni di segno distintivo dell'essere umano DC con una risposta infiammatoria sistemica moderata. Contrariamente al modello di ratto, un gran numero di modelli murini geneticamente modificati sullo sfondo C57BL/6 è disponibile. La combinazione di questi animali con il nostro modello consente i possibili ruoli di diverse citochine, chemochine o recettori di molecole bioattive (ad es., interleuchina (IL) -17; IL-10, chemokine CXCL13, recettori per le chemochine CXCR5 e CCR7 e il recettore della sfingosina-1-fosfato 4) deve essere valutata nella patogenesi della DC. La disponibilità di diversi ceppi di topi Congenici sullo sfondo C57BL/6 facilita in gran parte gli esperimenti di trasferimento per stabilire i ruoli dei tipi cellulari coinvolti nell'eziologia della DC. Infine, il modello offre l'opportunità di valutare le influenze di interventi locali (ad esempio, modifica del microbioma locale o terapia antinfiammatoria locale) su immunità mucosale nei segmenti intestinali affetti e non affetti e il su omeostasi immunitaria sistemica.

Chirurgia è ben tollerato sia nei gruppi di sham (colotomy) e sperimentale (colostomia). Mortalità perioperatoria non deve superare il 10% quando la chirurgia e perioperative management viene eseguita correttamente. Nella prima settimana postoperatoria, perdita di peso significativa è visto in entrambi i gruppi sperimentali e sham. Gli animali del gruppo finto raggiungono loro nadir di peso solitamente verso il quarto giorno postoperatorio, ma si verifica un giorno più tardi nel grupp...

Watch this Scientific Journal Video about Murine Distal Colostomy, A Novel Model of Diversion Colitis in C57BL/6 Mice at JoVE.com

Murine Distal Colostomy, A Novel Model of Diversion Colitis in C57BL/6 Mice

Murino colostomia distale, un nuovo modello di colite di diversione in topi C57BL/6

Murino colostomia distale fornisce un modello murino per colite di diversione umana, una colite linfocitaria prevalentemente nel segmento del colon escluso dal flusso fecale.

Diversion colitis (DC) is a frequent clinical condition occurring in patients with bowel segments excluded from the fecal stream as a result of a ...

murine-distal-colostomy-novel-model-diversion-colitis-c57bl6

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

12.8K Views.  Universitätsmedizin Greifswald. Murino colostomia distale fornisce un modello murino per colite di diversione umana, una colite linfocitaria prevalentemente nel segmento del colon escluso dal flusso fecale.

Video: Murine Distal Colostomy, A Novel Model of Diversion Colitis in C57BL/6 Mice

A Genetically Engineered Mouse Model of Sporadic Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, colorectal cancer mouse models based on tumor cell injection have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Genetically engineered mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in large organs such as the colon in which only a single tumor is desired.
As a result, an immunocompetent, genetically engineered mouse model of colorectal cancer was developed which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor development is initiated by surgical, segmental infection of the distal colon with adeno-cre virus in compound conditionally mutant mice. The tumors can be easily detected and monitored via colonoscopy. We here describe the surgical technique of segmental adeno-cre infection of the colon, the surveillance of the tumor via high-resolution colonoscopy and present the resulting colorectal tumors.

A protocol for the establishment of a genetically engineered mouse model of colorectal cancer by segmental adeno-cre infection and its surveillance via high-resolution colonoscopy is presented.

Un modello di mouse geneticamente modificato del cancro colorettale sporadico

Despite the advantages of easy applicability and cost-effectiveness, colorectal cancer mouse models based on tumor cell injection have severe limitations ...

Ricerca

Ricerca sul cancro

Modeling Colitis-Associated Cancer with Azoxymethane (AOM) and Dextran Sulfate Sodium (DSS)

Individuals with inflammatory bowel disease (IBD), such as Crohn's disease (CD) or ulcerative colitis (UC) are at increased risk of developing colorectal cancer (CRC) over healthy individuals. This risk is proportional to the duration and extent of disease, with a cumulative incidence as high as 30% in individuals with longstanding UC with widespread colonic involvement.1 Colonic dysplasia in IBD and colitis associated cancer (CAC) are believed to develop as a result of repeated cycles of epithelial cell injury and repair while these cells are bathed in a chronic inflammatory cytokine milieu.2 While spontaneous and colitis-associated cancers share the quality of being adenocarcinomas, the sequence of underlying molecular events is believed to be different.3 This distinction argues the need for specific animal models of CAC.
Several mouse models currently exist for the study of CAC. Dextran sulfate sodium (DSS), an agent with direct toxic effects on the colonic epithelium, can be administered in drinking water to mice in multiple cycles to create a chronic inflammatory state. With sufficient duration, some of these mice will develop tumors.4 Tumor development is hastened in this model if administered in a pro-carcinogenic setting. These include mice with genetic mutations in tumorigenesis pathways (APC, p53, Msh2), as well as mice pre-treated with genotoxic agents (azoxymethane [AOM], 1,2-dimethylhydrazine [DMH]).5 
The combination of DSS with AOM as a model for colitis associated cancer has gained popularity for its reproducibility, potency, low price, and ease of use. Though they have a shared mechanism, AOM has been found to be more potent and stable in solution than DMH. While tumor development in other models generally requires several months, mice injected with AOM and subsequently treated with DSS develop adequate tumors in as little as 7-10 weeks.6, 7 Finally, AOM and DSS can be administered to mice of any genetic background (knock out, transgenic, etc.) without cross-breeding to a specific tumorigenic strain. Here, we demonstrate a protocol for inflammation-driven colonic tumorigenesis in mice utilizing a single injection of AOM followed by three seven-day cycles of DSS over a 10 week period. This model induces tumors with histological and molecular changes closely resembling those occurring in human CAC and provides a highly valuable model for the study of oncogenesis and chemoprevention in this disease.8

Modeling Colitis-Associated Cancer with Azoxymethane AOM and Dextran Sulfate Sodium DSS

We demonstrate a protocol in which administration of the genotoxic agent azoxymethane (AOM) followed by three cycles of the pro-inflammatory agent dextran sulfate sodium (DSS) rapidly and consistently generates colon tumors in mice with morphologic and molecular similarities to those seen in human colitis-associated cancer.

Modellazione Colite-Associated Cancro con azoxymethane (OMA) e sodio solfato destrano (DSS)

Individuals with inflammatory bowel disease (IBD), such as Crohn's disease (CD) or ulcerative colitis (UC) are at increased risk of developing colorectal ...

Medicina

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

This protocol outlines the steps required to produce a robust model of infectious disease and colitis, as well as the methods used to characterize Citrobacter rodentium infection in mice. C. rodentium is a gram negative, murine specific bacterial pathogen that is closely related to the clinically important human pathogens enteropathogenic E. coli and enterohemorrhagic E. coli. Upon infection with C. rodentium, immunocompetent mice suffer from modest and transient weight loss and diarrhea. Histologically, intestinal crypt elongation, immune cell infiltration, and goblet cell depletion are observed. Clearance of infection is achieved after 3 to 4 weeks. Measurement of intestinal epithelial barrier integrity, bacterial load, and histological damage at different time points after infection, allow the characterization of mouse strains susceptible to infection.
The virulence mechanisms by which bacterial pathogens colonize the intestinal tract of their hosts, as well as specific host responses that defend against such infections are poorly understood. Therefore the C. rodentium model of enteric bacterial infection serves as a valuable tool to aid in our understanding of these processes. Enteric bacteria have also been linked to Inflammatory Bowel Diseases (IBDs). It has been hypothesized that the maladaptive chronic inflammatory responses seen in IBD patients develop in genetically susceptible individuals following abnormal exposure of the intestinal mucosal immune system to enteric bacteria. Therefore, the study of models of infectious colitis offers significant potential for defining potentially pathogenic host responses to enteric bacteria. C. rodentium induced colitis is one such rare model that allows for the analysis of host responses to enteric bacteria, furthering our understanding of potential mechanisms of IBD pathogenesis; essential in the development of novel preventative and therapeutic treatments.

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

Citrobacter rodentium infection provides a valuable model to study enteric bacterial infections as well as host immune responses and colitis in mice. This protocol outlines the measurement of barrier integrity, pathogen load and histological damage allowing for the thorough characterization of pathogen and host contributions to murine infectious colitis.

Il<em&gt; Citrobacter rodentium</em&gt; Mouse Model: Studiare Contributi degli agenti patogeni e Host alla colite infettiva

This protocol  outlines the steps required to produce a robust model of infectious disease and  colitis, as well as the methods used to characterize ...

Immunologia e infezioni

Flexible Colonoscopy in Mice to Evaluate the Severity of Colitis and Colorectal Tumors Using a Validated Endoscopic Scoring System

The use of modern endoscopy for research purposes has greatly facilitated our understanding of gastrointestinal pathologies. In particular, experimental endoscopy has been highly useful for studies that require repeated assessments in a single laboratory animal, such as those evaluating mechanisms of chronic inflammatory bowel disease and the progression of colorectal cancer. However, the methods used across studies are highly variable. At least three endoscopic scoring systems have been published for murine colitis and published protocols for the assessment of colorectal tumors fail to address the presence of concomitant colonic inflammation. This study develops and validates a reproducible endoscopic scoring system that integrates evaluation of both inflammation and tumors simultaneously. This novel scoring system has three major components: 1) assessment of the extent and severity of colorectal inflammation (based on perianal findings, transparency of the wall, mucosal bleeding, and focal lesions), 2) quantitative recording of tumor lesions (grid map and bar graph), and 3) numerical sorting of clinical cases by their pathological and research relevance based on decimal units with assigned categories of observed lesions and endoscopic complications (decimal identifiers). The video and manuscript presented herein were prepared, following IACUC-approved protocols, to allow investigators to score their own experimental mice using a well-validated and highly reproducible endoscopic methodology, with the system option to differentiate distal from proximal endoscopic colitis (D-PECS).

Over the past few years, new generation endoscopes have emerged as important diagnostic research aids for evaluating murine colitis and colorectal tumors. We present herein a detailed protocol for endoscopic assessment of inflammation and colorectal tumors in mice, as well as a novel scoring system that uses decimal identifiers to document the endoscopic severity of colitis and colorectal tumors.

La colonscopia flessibile in topi per valutare la gravità della colite e colon-retto Tumori Utilizzo di un sistema Validated endoscopica Scoring

The use of modern endoscopy for research purposes has greatly facilitated our understanding of gastrointestinal pathologies. In particular, experimental ...

Murine Colitis Modeling using Dextran Sulfate Sodium (DSS)

Colitis can occur from viral or bacterial infections, ischemic insult, or autoimmune disorders; most notably Ulcerative Colitis and the colonic variant of Crohn&rsquo;s Disease - Crohn&rsquo;s Colitis. Acute colitis may present with abdominal pain and distention, malabsorption, diarrhea, hematochezia and mucus in the stool. We are beginning to understand the complex interactions between the environment, genetics, and epithelial barrier dysfunction in Inflammatory Bowel Disease and animal models of colitis have been essential in advancing our understanding of this disease. One popular model involves supplementing the drinking water of mice with low-molecular weight Dextran Sodium Sulfate (DSS), resulting in epithelial damage and a robust inflammatory response in the colon lasting several days 1.Variations of this approach can be used to model acute injury, acute injury followed by repair, and repeated cycles of DSS interspersed with recovery modeling chronic inflammatory diseases 2. After a single four-day treatment of 3% DSS in drinking water, mice show signs of acute colitis including weight loss, bloody stools, and diarrhea. Mice are euthanized at the conclusion of the treatment course and at necropsy dissected colons are processed and can be 'Swiss rolled" 3 to allow microscopic analysis of the entire colon or infused with formalin as "sausages" to allow macroscopic analysis. Tissue is then embedded in paraffin, sectioned, and stained for histologic review.

Murine Colitis Modeling using Dextran Sulfate Sodium DSS

Dextran sulfate sodium (DSS) administered in the drinking water is an established murine inflammatory injury model of acute colitis. This protocol outlines the method for DSS treatment and the preparation of tissues.

Colite Modeling murino utilizzando destrano sodio solfato (DSS)

Colitis can occur from viral or bacterial infections, ischemic insult, or autoimmune disorders; most notably Ulcerative Colitis and the colonic variant of ...

Biologia

Creation of Colonic Anastomosis in Mice

Intestinal anastomoses are commonly performed in both elective and emergent operations. Even so, anastomotic leaks are a highly feared complications of colonic surgeries and can occur in up to 26% of surgical anastomoses, with mortality being up to 39% for patients with such a leak. Currently, there remains a paucity of data detailing the cellular mechanisms of anastomotic healing. Devising preventative strategies and treatment modalities for anastomotic leak could be greatly potentiated by a better understanding of appropriate anastomotic healing.
A murine model is ideal as previous studies have shown that the murine anastomosis is the most clinically similar to the human case as compared with other animal models. We offer an easily reproducible murine model of colonic anastomosis in mice that will allow for further illustration of anastomotic healing.

Anastomotic leak or breakdown after surgery is a major cause of postoperative morbidity and mortality. Our surgery for creating a colonic anastomosis is a reliable and reproducible method for studying the healing mechanisms of anastomoses.

Creazione dell'anastomosi colica in topi

Intestinal anastomoses are commonly performed in both elective and emergent operations. Even so, anastomotic leaks are a highly feared complications of ...

Murine Appendectomy Model of Chronic Colitis Associated Colorectal Cancer by Precise Localization of Caecal Patch

The human appendix has been recently implicated to play important biological roles in the pathogenesis of various complex diseases, such as colorectal cancer, inflammatory bowel disease, and Parkinson&#8217;s disease. To study the function of the appendix, a gut disease-associated murine appendectomy model has been established and its step-by-step protocol is described here. This report introduces a facile protocol for caecal patch removal in mice followed by the chemical induction of chronic colitis-associated colorectal cancer using a combination of dextran sulfate sodium (DSS) and azoxymethane (AOM). IgA specific cells and IgA concentration were significantly reduced upon removal of the caecal patch in male C57BL/6 mice&#160;compared to those in the sham group. Simultaneously administering 2% DSS and AOM resulted in nearly 80% mice survival in both sham and appendectomy groups without significant body weight loss. Histological results confirmed colonic inflammation and different degrees of adenocarcinoma. This model can be used for the study of the functional role of the appendix in maintaining gut microbiota homeostasis and pathogenesis of gut colitis and malignancies, as well as for the potential development of drug targeting therapies.

The presented protocol describes a facile surgical removal of the appendix (caecal patch) in a mouse followed by the induction of inflammatory bowel disease-associated colorectal cancer. This murine appendectomy model enables investigation of the biological role of the appendix in the pathogenesis of human gastrointestinal disease.

Murine Appendectomy Modello di Cronica Colite Associata Cancro Colorettale da Localizzazione precisa di Patch Caecal

The human appendix has been recently implicated to play important biological roles in the pathogenesis of various complex diseases, such as colorectal ...

Single-Anastomosis Duodeno-Ileal Bypass with Sleeve Gastrectomy Model in Mice

Obesity is a major health issue worldwide. As a response, bariatric surgeries have emerged to treat obesity and its related comorbidities (e.g., diabetes mellitus, dyslipidemia, non-alcoholic steatohepatitis, cardiovascular events, and cancers) through restrictive and malabsorptive mechanisms. Understanding the mechanisms by which these procedures allow such improvements often require their transposition into animals, especially in mice, because of the ease of generating genetically modified animals. Recently, the single-anastomosis duodeno-ileal bypass with sleeve gastrectomy (SADI-S) has emerged as a procedure that uses both restrictive and malabsorptive effects, which is being used as an alternative to gastric bypass in case of major obesity. Thus far, this procedure has been associated with strong metabolic improvements, which has led to a marked increase in its use in daily clinical practice. However, the mechanisms underlying these metabolic effects have been poorly studied as a result of a lack of animal models. In this article, we present a reliable and reproducible model of SADI-S in mice, with a special focus on perioperative management. The description and use of this new rodent model will be helpful for the scientific community to better understand the molecular, metabolic, and structural changes induced by the SADI-S and to better define the surgical indications for clinical practice.

Single-anastomosis duodeno-ileal bypass (SADI-S) is an emerging bariatric procedure with important metabolic effects. In this article, we present a reliable and reproducible model of SADI-S in mice.

Bypass duodeno-ileale a singola anastomosi con modello di gastrectomia a manica nei topi

Obesity is a major health issue worldwide. As a response, bariatric surgeries have emerged to treat obesity and its related comorbidities (e.g., diabetes ...

A Mouse Model of Intestinal Partial Obstruction

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including tumorous growths. However, the cellular remodeling mechanisms involved in, and caused by, intestinal obstructions are poorly understood. Several animal models of intestinal obstructions have been developed, but the mouse model is the most cost/time effective. The mouse model uses the surgical implantation of an intestinal partial obstruction (PO) that has a high mortality rate if it is not performed correctly. In addition, mice receiving PO surgery fail to develop hypertrophy if an appropriate blockade is not used or not properly placed. Here, we describe a detailed protocol for PO surgery which produces reliable and reproducible intestinal obstructions with a very low mortality rate. This protocol utilizes a surgically placed silicone ring that surrounds the ileum which partially blocks digestive movement in the small intestine. The partial blockage makes the intestine become dilated due to the halt of digestive movement. The dilation of the intestine induces smooth muscle hypertrophy on the oral side of the ring that progressively develops over 2 weeks until it causes death. The surgical PO mouse model offers an in vivo model of hypertrophic intestinal tissue useful for studying pathological changes of intestinal cells including smooth muscle cells (SMC), interstitial cells of Cajal (ICC), PDGFR&#945;+, and neuronal cells during the development of intestinal obstruction.

Intestinal obstructions are a partial or complete blockage of the intestine that can cause severe abdominal pain, nausea, vomiting, and preventing the passage of stool. This procedure for creating intestinal partial obsructions in mice is reliable in studying the mechanisms underlying pathological cell growth and death in the intestine.

Un modello murino di ostruzione intestinale parziale

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including ...

A Technique to Generate a Murine Model of Dextran Sulfate Sodium-Induced Colitis

Source: Du, Y., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/58668">Effects of Taste Signaling Protein Abolishment on Gut Inflammation in an Inflammatory Bowel Disease Mouse Model.</a> J. Vis. Exp. (2018)
This video demonstrates a technique to generate a chemical-induced murine model of colitis &#8212; an inflammatory bowel disease. The mouse is allowed to consume a dextran sodium sulfate (DSS) solution for an adequate duration. Upon consumption, DSS damages the mucus and epithelial layer barrier in the colon, allowing gut microbes to enter the underlying tissue &#8212; where they are sensed by the resident immune cells to mediate an exaggerated immune response, resulting in inflammation.

Una tecnica per generare un modello murino di colite indotta da destrano solfato di sodio

Source: Du, Y., et al. Effects of Taste Signaling Protein Abolishment on Gut Inflammation in an Inflammatory Bowel Disease Mouse Model. J. Vis. Exp. ...