The evolution of the device started with somewhat different cutting prototype demonstrated at the Imperial Cancer Research Fund (ICRF) mouse club by Bass Hassan, following this there were several discussions with Rob Rudling and Nikki Mandir in the containment facility at ICRF Clare Hall. Jo Kitau of the mechanical workshop at ICRF constructed the initial versions and Andy Lucas designed and perfected the final model in the CRUK workshop in Oxford.

Le varie procedure sperimentali e l&#39;eutanasia umana sono approvati dal comitato etico istituzioni ospitanti e dai competenti organismi di regolamentazione. L&#39;eutanasia è normalmente realizzato utilizzando CO 2 asfissia seguita da dislocazione cervicale. 1. Dispositivo <ol><li> Macchina la porzione principale della base del dispositivo di un blocco solido di alta densità resina acetalica, come descritto in questo manoscritto. A ciascuna estremità creare 3 mm strisce alti elevati, e la macchina parte da questi 2,5 mm boschetti per prendere le aste (vedi Figura 2 e Figura 3). </li><li> Fai barre di acciaio inossidabile di diametro 2,4 millimetri con estremità arrotondate-off. NOTA: Il telaio principale del coperchio è stata lavorata su piastra duralluminio. Un numero limitato di dispositivi finiti sono disponibili presso Dr. Goodlad. </li></ol> 2. Uso del dispositivo <ol><li> Sezionare l&#39;intestino tenue e del colon dalla cavità addominale con le forbicie le dita guantate. Rimuovere il mesentere tenendola con pinze curve e delicatamente tirando via dall&#39;intestino. </li><li> Sciacquare l&#39;intestino con fosfato freddo salina tamponata (PBS, pH 7,4) con un puntale tipo Gilson, che era stato abbattuto alla sua estremità più larga in modo che possa andare bene su un 10 o 20 ml luer siringa di plastica. </li><li> Disporre il piccolo intestino (SB) su un foglio di carta assorbente e dividere in tre parti uguali di lunghezza (prossimale, medio e distale - SB1, SB2 e SB3) con le forbici. Rimuovere il cieco e il layout del colon. </li><li> Fai diverse molto piccoli tagli nelle segmenti intestinali con le forbici per consentire al fluido di fuggire e mettere un foglio di carta assorbente sopra l&#39;intestino e delicatamente eseguire un dito sopra i preparativi per spremere il liquido e asciugare. </li><li> Sollevare le sezioni e inserire le barre di acciaio inossidabile che in precedenza erano bagnati immergendo in un bicchiere di tampone fosfato. NOTA: L&#39;intestino può essereconvertito se lo si desidera (rovesciato). </li><li> Etichettare un pezzo di carta o un filtro di carta tagliata a misura per adattarsi alla base del dispositivo. Pencil è migliore, in quanto non è influenzato da praticamente tutte le soluzioni. Data Autopsia, codice sperimentale ed il numero di identificazione degli animali sono suggeriti. </li><li> Posizionare la carta o carta da filtro nella base del dispositivo. Inserire le aste e gli intestini nelle fessure alla base del dispositivo. Assicurarsi che l&#39;estremità prossimale dei segmenti è posizionato in maniera standard. Le estremità prossimali devono essere vicino le etichette di carta. </li><li> Posizionare la parte superiore del dispositivo sopra la base. Utilizzare le barre angolari per guidare una lama di bisturi per tagliare longitudinalmente sezioni. NOTA: Aiuta se il tessuto è delicatamente e accuratamente tenuto con un dito per garantire che non si muove con il coltello. </li><li> Rimuovere la parte superiore del dispositivo. Rimuovere con cura la carta e tessuti di filtro (ancora sulla loro aste). NOTA: Un pezzo di carta rigida posto sotto il filcarta ter aiuterà sollevamento. </li><li> Leggermente umido (con un dito guantato immerso in PBS) i segmenti. Arrotolare la verga, lato a lato per aprire l&#39;intestino e diffonderla piatta. Il tessuto viene quindi aderire alla carta da filtro. </li><li> Esaminare visivamente i preparativi per eventuali lesioni macroscopiche. </li><li> Trasferire il tessuto aderito alla carta da filtro per un bagno poco profondo (o casella di sandwich) contenente fissativo. NOTA: Dopo il tessuto aderisce alla carta da filtro o di carta rimarrà saldamente collegato. Una vasta gamma di fissativi può essere utilizzata per mantenere la preparazione. Si raccomanda che il liquido di Carnoy dovrebbe essere utilizzato per lo studio delle proliferazioni cellulari intestinali 6, che è la fissazione ideale per i migliori metodi di proliferazione cellulare; tuttavia formalina o molti altri fissativi possono essere utilizzati anche se sono necessari altri endpoint. </li><li> Dopo la fissazione (solitamente 3 ore), trasferire i preparativi in ​​etanolo al 70%. I campioni possono essere conservati fino al momento. Le preparazioni cun essere studiati en face e / o utilizzati per la preparazione di campioni istologici per H &amp; E o altre tecniche di colorazione istologiche, immunoistochimica e / o ibridazione in situ. NOTA: fissazione prolungata può ridurre la reattività immunoistochimica e / o ibridazione in situ reattività. Scatole panino polipropilene sono ideali per la memorizzazione di un gran numero di preparazioni. </li><li> Se necessario i tessuti fissati dai vari segmenti intestinali può essere fatto in &#39;rotoli svizzera e colorate con l&#39;intera gamma di metodi istologici 6 compresi quelli sopra descritti. Per fare un &#39;Swiss roll&#39; i segmenti intestinali vengono rimossi dalla carta da filtro, arrotolata intorno ad un bastoncino da cocktail o uno stuzzicadenti, fissato con un perno entomologico e trattati a cera incorporamento 6. </li><li> Se necessario, macchia il tessuto all&#39;ingrosso con blu di metilene di visualizzare aberranti cripta foci 7. </li><li> Punteggio preparativi fissatitempo libero. </li></ol>

<ol><li>Goodlad, R. A. Cancer Handbook. Alison, M. R. , John Wiley and Sons, Ltd. 243-262 (2007).</li>
<li>Ward, J. M., Treuting, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rodent+intestinal+epithelial+carcinogenesis%3A+pathology+and+preclinical+models%5BTitle%5D)+AND+%22Toxicol+Pathol%22%5BJournal%5D)">Rodent intestinal epithelial carcinogenesis: pathology and preclinical models.</a> Toxicol Pathol. 42, 148-161 (2014).</li>
<li>Sundberg, J. P., Hogenesch, H., Nikitin, A. Y., Treuting, P. M., Ward, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Training+mouse+pathologists%3A+ten+years+of+workshops+on+the+Pathology+of+Mouse+Models+of+Human+Disease%5BTitle%5D)+AND+%22Toxicol+Pathol%22%5BJournal%5D)">Training mouse pathologists: ten years of workshops on the Pathology of Mouse Models of Human Disease.</a> Toxicol Pathol. 40, 823-825 (2012).</li>
<li>Wasan, H. S., Novelli, M., Bee, J., Bodmer, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dietary+fat+influences+on+polyp+phenotype+in+multiple+intestinal+neoplasia+mice%5BTitle%5D)+AND+%22Proc+Natl+Acade+Sci+USA%22%5BJournal%5D)">Dietary fat influences on polyp phenotype in multiple intestinal neoplasia mice.</a> Proc Natl Acade Sci USA. 94, 3308-3313 (1997).</li>
<li>Rudling, R., Hassan, A. B., Kitau, J., Mandir, N., Goodlad, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+simple+device+to+rapidly+prepare+whole+mounts+of+murine+intestine%5BTitle%5D)+AND+%22Cell+Prolif%22%5BJournal%5D)">A simple device to rapidly prepare whole mounts of murine intestine.</a> Cell Prolif. 39, 415-420 (2006).</li>
<li>Alferez, D., Goodlad, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((To+best+measure+cell+proliferation+in+samples+from+the+intestine%5BTitle%5D)+AND+%22Cell+Prolif%22%5BJournal%5D)">To best measure cell proliferation in samples from the intestine.</a> Cell Prolif. 40, 231-240 (2007).</li>
<li>Park, H. S., Goodlad, R. A., Wright, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+incidence+of+aberrant+crypt+foci+and+colonic+carcinoma+in+dimethylhydrazine-treated+rats+varies+in+a+site-specific+manner+and+depends+on+tumor+histology%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">The incidence of aberrant crypt foci and colonic carcinoma in dimethylhydrazine-treated rats varies in a site-specific manner and depends on tumor histology.</a> Cancer Res. 57, 4507-4510 (1997).</li>
<li>Park, H. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+epidermal+growth+factor+and+dimethylhydrazine+on+crypt+size%2C+cell+proliferation%2C+and+crypt+fission+in+the+rat+colon.+Cell+proliferation+and+crypt+fission+are+controlled+independently%5BTitle%5D)+AND+%22Am+J+Pathol%22%5BJournal%5D)">Effects of epidermal growth factor and dimethylhydrazine on crypt size, cell proliferation, and crypt fission in the rat colon. Cell proliferation and crypt fission are controlled independently.</a> Am J Pathol. 151, 843-852 (1997).</li>
<li>Femia, A. P., Dolara, P., Caderni, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mucin-depleted+foci+%28MDF%29+in+the+colon+of+rats+treated+with+azoxymethane+%28AOM%29+are+useful+biomarkers+for+colon+carcinogenesis%5BTitle%5D)+AND+%22Carcinogenesis%22%5BJournal%5D)">Mucin-depleted foci (MDF) in the colon of rats treated with azoxymethane (AOM) are useful biomarkers for colon carcinogenesis.</a> Carcinogenesis. 25, 277-281 (2004).</li>
<li>Ruehl-Fehlert, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Revised+guides+for+organ+sampling+and+trimming+in+rats+and+mice--part+1%5BTitle%5D)+AND+%22Exp+Toxicol+Pathol%22%5BJournal%5D)">Revised guides for organ sampling and trimming in rats and mice--part 1.</a> Exp Toxicol Pathol. 55, 91-106 (2003).</li>
<li>Goodlad, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inhibiting+vascular+endothelial+growth+factor+receptor-2+signaling+reduces+tumor+burden+in+the+ApcMin%2F%2B+mouse+model+of+early+intestinal+cancer%5BTitle%5D)+AND+%22Carcinogenesis%22%5BJournal%5D)">Inhibiting vascular endothelial growth factor receptor-2 signaling reduces tumor burden in the ApcMin/+ mouse model of early intestinal cancer.</a> Carcinogenesis. 27, 2133-2139 (2006).</li>
<li>Mandir, N., Englyst, H., Goodlad, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Resistant+carbohydrates+stimulate+cell+proliferation+and+crypt+fission+in+wild-type+mice+and+in+the+Apc%28Min%2F%2B%29+mouse+model+of+intestinal+cancer%2C+association+with+enhanced+polyp+development%5BTitle%5D)+AND+%22Br+J+Nutr%22%5BJournal%5D)">Resistant carbohydrates stimulate cell proliferation and crypt fission in wild-type mice and in the Apc(Min/+) mouse model of intestinal cancer, association with enhanced polyp development.</a> Br J Nutr. 100, 711-721 (2008).</li>
</ol>

The authors have nothing to disclose.

Il dispositivo accelera notevolmente il processo in modo che due operatori possono autopsia completamente un mouse all&#39;interno 6 min. Ciò includerà un controllo visivo per lesioni macroscopiche evidenti e la pesatura e alla fissazione degli organi principali. Essa consentirà inoltre agli operatori di lavare, asciugare e pesare lo stomaco, intestino cieco, intestino tenue e del colon e preparare l&#39;intero monti. Il tessuto appiattita può essere fissato molto rapidamente e così evitare artefatti degradazione. ...

Il mouse e il topo geneticamente modificato 1-3 sono un modello prezioso per lo studio di diverse patologie soprattutto tumorali e il suo rapporto infiammazione nel tratto gastrointestinale. Questo spesso richiede la preparazione di supporti interi del piccolo intestino e / o del colon. La preparazione di supporti integrali può essere fatto con un paio di forbici precisione di offset ma può richiedere circa 20 minuti per topo 4. Questo non è molto pratico per studi comparativi, che richiedono una notevole quantità di topi (dire 10 per gruppo in analisi statistica). Inoltre, il tempo necessario aumenta il rischio di degradazione dei tessuti. Un tentativo di alleviare questo problema ha portato alla sperimentazione di vari aiuti di preparazione taglio. Il primo era basato su una serie di piastre metalliche, con alberi di taglio, ma i piatti oscurata il tessuto e il taglio era così difficile da controllare. Un conventional matita sei facce finalmente fornito l&#39;ispirazione per il dispositivo di taglio, come abbiamo potuto poi vedere come una forma di mezza matita sarebbe una buona guida per un bisturi (Figura 1). Ciò ha provocato un disegno per un telaio con quattro guide di taglio 5. L&#39;efficacia di questo dispositivo era drammatica come le preparazioni in Figura 4 mostrano la differenza tra la preparazione e la preparazione forbice dispositivo. Le cime dei dispositivi originali è stato costruito da diversi pezzi di metallo, ma quelli successivi, e il modello attuale è stata lavorata da un solido pezzo di duralluminio. La base è stata lavorata su qualità resina ad alta densità. Il dispositivo ha dimostrato di essere di grande utilità per la preparazione di supporti interi di topo dell&#39;intestino in quanto accelera notevolmente e migliora la qualità e la consistenza delle preparazioni, che hanno una vasta gamma di applicazioni.

<table><tbody><tr><td>Delrin</td><td>Ryan Plastics, Earls Barton, UK</td><td></td><td>High-density acetal resin similar material would suffice</td></tr><tr><td>Duralumin plate</td><td>Metal Supplies Ltd, Park Road,Dukinfield. SK16 5LP UK</td><td></td><td>Aluminium allow dating back to 1909 so alterative suppliers are available&amp;nbsp;</td></tr><tr><td>Finished device(s) ready for use</td><td>Contact Dr Goodlad&amp;nbsp; r.goodlad@imperial.ac.uk</td><td></td><td>Contact&amp;nbsp; r.goodlad@imperial.ac.uk for supply details</td></tr></tbody></table>

a simple device to rapidly prepare whole mounts of the mouse intestine

Preparazione supporti interi dell&#39;intestino tenue e del colon del mouse per la successiva analisi o la quantificazione può essere lunga e difficile. Descriviamo l&#39;uso di un dispositivo semplice da tagliare e intestini &#39;roll&#39; del mouse per preparare rapidamente intere preparazioni montaggio di qualità superiore ed uniforme a quello che può essere ottenuto manualmente. Il dispositivo comprende una base che contiene 4 barre in acciaio inox e una parte superiore, che funge da guida per il taglio. Le aste sono inseriti nel lume dell&#39;intestino tenue [diviso in terzi] e il colon. Le aste ed i campioni vengono poi poste su un pezzo di carta da filtro o carta nelle scanalature di tenuta nella base del dispositivo. La parte superiore del dispositivo viene quindi posizionato e funge da guida per il taglio. Le due sezioni ad angolo al centro del pezzo superiore sono utilizzati per guidare un coltello o bisturi e tagliare gli intestini longitudinalmente sulla parte superiore delle aste. Una volta che le sezioni intestinali sono stati tagliati, la parte superiore viene rimosso e la carta, tissue e aste delicatamente rimosso dal dispositivo e posto in panchina. Le aste sono poi delicatamente arrotolate lateralmente per appiattire e incollare i segmenti intestinali sul pezzo di carta da filtro sottostante o carta. La preparazione finale può essere esaminato o fisso e memorizzati per una successiva analisi. I preparativi sono preziosi per lo studio dei cambiamenti intestinali nei modelli normali o geneticamente modificati mouse. I preparativi sono stati utilizzati per lo studio e la quantificazione degli effetti di infiammazione (colite), danni, lesioni pre-cancerose (aberranti crypt foci (ACFS) e Mucin impoverito focolai (MDF)) e polipi o tumori.

Polipi sono facili da identificare sotto uno stereomicroscopio con fonte luminosa all&#39;altra illuminare il tessuto (Figura 4). E &#39;utile per segnare la carta con segni di matita quando un polipo si osserva in modo che punteggio può essere discusso più tardi e diversi operatori in grado di raggiungere un accordo. Il tessuto può anche essere massa macchiato [con blu di metilene] per identificare aberranti foci 8 o mucina foci impoverito (MDF) 9 dall&#39;illuminazione dall&#3...

Watch this Scientific Journal Video about A Simple Device to Rapidly Prepare Whole Mounts of the Mouse Intestine at JoVE.com

A Simple Device to Rapidly Prepare Whole Mounts of the Mouse Intestine

The use of a simple device to cut and &#8216;roll&#8217; mouse intestines to rapidly prepare whole mount preparations is described.

Un dispositivo semplice da preparare rapidamente intere Monti della Intestino mouse

Preparing whole mounts of the mouse small intestine and colon for subsequent analysis or quantification can be time consuming and difficult. We describe ...

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Research

JoVE Journal

Medicine

19.6K Views.  National Institutes of Health. The use of a simple device to cut and ‘roll’ mouse intestines to rapidly prepare whole mount preparations is described.

Video: A Simple Device to Rapidly Prepare Whole Mounts of the Mouse Intestine

Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models

The epithelial surface of the mammalian intestine is a dynamic tissue that renews every 3 - 7 days. Understanding this renewal process identified a population of rapidly cycling intestinal stem cells (ISCs) characterized by their expression of the Lgr5 gene. These are supported by a quiescent stem cell population, marked by Bmi-1 expression, capable of replacing them in the event of injury. Investigating the interactions between these populations is crucial to understanding their roles in disease and cancer. The ISCs exist within crypts on the intestinal surface, these niches support the ISC in replenishing the epithelia. The interaction between active and quiescent ISCs likely involves other differentiated cells within the niche, as it has previously been demonstrated that the &#8216;&#8216;stemness&#8217;&#8217; of the Lgr5 ISC is closely tied to the presence of their neighboring Paneth cells. Using conditional cre-lox mouse models we tested the effect of deleting the majority of active ISCs in the presence or absence of the Paneth cells. Here we describe the techniques and analysis undertaken to characterize the intestine and demonstrate that the Paneth cells play a crucial role within the ISC niche in aiding recovery following substantial insult.

Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models

Intestinal epithelial stem cells (ISCs) are intermingled with Paneth cells. These cells are differentiated progeny of the ISC, which support the ISCs and provide antibacterial protection. Here we demonstrate how we used transgenic conditional mouse models to establish that Paneth cells play a crucial role in maintaining the intestinal epithelia.

Protocolli per l&#39;analisi del ruolo delle cellule Paneth in Rigenerazione della murino Intestino con condizionale<em&gt; Cre-lox</em&gt; Modelli mouse

The epithelial surface of the mammalian intestine is a dynamic tissue that renews every 3 - 7 days. Understanding this renewal process identified a ...

Ricerca

Biologia dello sviluppo

Functional Assessment of Intestinal Motility and Gut Wall Inflammation in Rodents: Analyses in a Standardized Model of Intestinal Manipulation

Inflammation of the gastrointestinal tract is a common reason for a variety of human diseases. Animal research models are critical in investigating the complex cellular and molecular of intestinal pathology. Although the tunica mucosa is often the organ of interest in many inflammatory diseases, recent works demonstrated that the muscularis externa (ME) is also a highly immunocompetent organ that harbours a dense network of resident immunocytes.1,2 These works were performed within the standardized model of intestinal manipulation (IM) that leads to inflammation of the bowel wall, mainly limited to the ME. Clinically this inflammation leads to prolonged intestinal dysmotility, known as postoperative ileus (POI) which is a frequent and unavoidable complication after abdominal surgery.3 The inflammation is characterized by liberation of proinflammatory mediators such as IL-64 or IL-1&beta; or inhibitory neurotransmitters like nitric oxide (NO).5 Subsequently, tremendous numbers of immunocytes extravasate into the ME, dominated by polymorphonuclear neutrophils (PMN) and monocytes and finally maintain POI.2 Lasting for days, this intestinal paralysis leads to an increased risk of aspiration, bacterial translocation and infectious complications up to sepsis and multi organ failure and causes a high economic burden.6
In this manuscript we demonstrate the standardized model of IM and in vivo assessment of gastrointestinal transit (GIT) and colonic transit. Furthermore we demonstrate a method for separation of the ME from the tunica mucosa followed by immunological analysis, which is crucial to distinguish between the inflammatory responses in these both highly immunoactive bowel wall compartments. All analyses are easily transferable to any other research models, affecting gastrointestinal function.

Postoperative ileus (POI) is a complication of abdominal surgery leading to increased morbidity and a prolonged hospital stay. Because prophylactic or therapeutic strategies are lacking intensified research is necessary. Therefore we established a standardized and feasible mouse model to investigate the pathophysiology of POI and to study potential therapeutic options.

Valutazione funzionale della motilità intestinale e Wall infiammazione intestinale nei roditori: Analisi in un modello standardizzato di manipolazione intestinale

Inflammation of the gastrointestinal tract is a common reason for a variety of human diseases. Animal research models are critical in investigating the ...

Medicina

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological questions in cell and developmental biology. In addition, organoids together with recent technical advances in gene editing and viral gene delivery promises to advance medical research and development of new drugs for treatment of diseases. Organoids grown in vitro in basement matrix provide powerful model systems for studying the behavior and function of various proteins and are well suited for live-imaging of fluorescent-tagged proteins. However, establishing the expression and localization of the endogenous proteins in ex vivo tissue and in in vitro organoids is important to verify the behavior of the tagged proteins. To this end we have developed and modified tissue isolation, fixation, and immuno-labeling protocols for localization of microtubules, centrosomal, and associated proteins in ex vivo intestinal tissue and in in vitro intestinal organoids. The aim was for the fixative to preserve the 3D architecture of the organoids/tissue while also preserving antibody antigenicity and enabling good penetration and clearance of fixative and antibodies. Exposure to cold depolymerizes all but stable microtubules and this was a key factor when modifying the various protocols. We found that increasing the ethylenediaminetetraacetic acid (EDTA) concentration from 3 mM to 30 mM gave efficient detachment of villi and crypts in the small intestine while 3 mM EDTA was sufficient for colonic crypts. The developed formaldehyde/methanol fixation protocol gave very good structural preservation while also preserving antigenicity for effective labeling of microtubules, actin, and the end-binding (EB) proteins. It also worked for the centrosomal protein ninein although the methanol protocol worked more consistently. We further established that fixation and immuno-labeling of microtubules and associated proteins could be achieved with organoids isolated from or remaining within the basement matrix.

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

We present protocols for isolation of intestinal 3D structures from in vivo tissue and in vitro basement matrix embedded organoids, and detail different fixation and staining protocols optimized for immuno-labeling of microtubule, centrosomal, and junctional proteins as well as cell markers including the stem cell protein Lgr5.

Immuno-fluorescenza etichettatura dei microtubuli e proteine centrosomali in tessuto intestinale Ex Vivo e 3D In Vitro intestinale Organoids

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological ...

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the complexities of the organoid growth characteristics carry significant caveats for the investigator. Specifically, the growth patterns of each individual organoid are highly variable and create a heterogeneous population of epithelial cells in culture. With such caveats, common tissue culture practices cannot be simply applied to the organoid system due to the complexity of the cellular structure. Counting and plating based solely on cell number, which is common for individually separated cells, such as cell lines, is not a reliable method for organoids unless some normalization technique is applied. Normalizing to total protein content is made complex due to the resident protein matrix. These characteristics in terms of cell number, shape and cell type should be taken into consideration when evaluating secreted contents from the organoid mass. This protocol has been generated to outline a simple procedure to culture and treat small intestinal organoids with microbial pathogens and pathogen associated molecular patterns (PAMPs). It also emphasizes the normalization techniques that should be applied when protein analysis are conducted after such a challenge.

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Here, a protocol to harvest, maintain, and treat mouse small intestinal organoids with pathogen associated molecular patterns (PAMPs) and Listeria monocytogenes is described, as well as emphasis on gene expression and proper normalization techniques for protein.

Il<em&gt; Ex Vivo</em&gt; Cultura e Pattern Recognition recettore stimolazione del mouse intestinale organoidi

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the ...

Immunologia e infezioni

Visualization of the Interstitial Cells of Cajal (ICC) Network in Mice

The interstitial cells of Cajal (ICC) are mesenchymal derived "pacemaker cells" of the gastrointestinal (GI) tract that generate spontaneous slow waves required for peristalsis and mediate neuronal input from the enteric nervous system1. Different subtypes of ICC form distinct networks in the muscularis of the GI tract 2,3. Loss or injury to these networks is associated with a number of motility disorders4. ICC cells express the KIT receptor tyrosine kinase on the plasma membrane and KIT immunostaining has been used for the past 15 years to label the ICC network5,6. Importantly, normal KIT activity is required for ICC development5,6. Neoplastic transformation of ICC cells results in gastrointestinal stromal tumor (GIST), that frequently harbor gain-of-function KIT mutations7,8. We recently showed that ETV1 is a lineage-specific survival factor expressed in the ICC/GIST lineage and is a master transcriptional regulator required for both normal ICC network formation and for of GIST tumorigenesis9. We further demonstrate that it cooperates with activating KIT mutations in tumorigenesis. Here, we describe methods for visualization of ICC networks in mice, largely based on previously published protocols10,11. More recently, the chloride channel anoctamin 1 (ANO1) has also been characterized as a specific membrane marker of ICC11,12. Because of their plasma membrane localization, immunofluorescence of both proteins can be used to visualize the ICC networks. Here, we describe visualization of the ICC networks by fixed-frozen cyrosections and whole mount preparations.

Visualization of the Interstitial Cells of Cajal ICC Network in Mice

The interstitial cells of Cajal (ICC) are the pacemaker cells of the gastrointestinal (GI) tract. They form complex networks between smooth muscle cells and post-ganglionic neuronal fibers to regulate GI contractility. Here, we present immunofluorescence methods cross-sectional and whole-mount visualization of murine ICC networks.

Visualizzazione delle cellule interstiziali di Cajal (ICC) di rete in topi

The interstitial cells of Cajal (ICC) are mesenchymal derived "pacemaker cells" of the gastrointestinal (GI) tract that generate spontaneous slow waves ...

Biologia

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over developmental stages. Three-dimensional information from thin serial sections can be challenging to interpret because of the difficulty of reconstructing serial sections perfectly and maintaining proper orientation of the tissue over serial sections. Recent findings by Grosse et al., 2011 highlight the importance of three- dimensional information in understanding morphogenesis of the developing villi of the intestine1. Three-dimensional reconstruction of singly labeled intestinal cells demonstrated that the majority of the intestinal epithelial cells contact both the apical and basal surfaces. Furthermore, three-dimensional reconstruction of the actin cytoskeleton at the apical surface of the epithelium demonstrated that the intestinal lumen is continuous and that secondary lumens are an artifact of sectioning. Those two points, along with the demonstration of interkinetic nuclear migration in the intestinal epithelium, defined the developing intestinal epithelium as a pseudostratified epithelium and not stratified as previously thought1. The ability to observe the epithelium three-dimensionally was seminal to demonstrating this point and redefining epithelial morphogenesis in the fetal intestine. With the evolution of multi-photon imaging technology and three-dimensional reconstruction software, the ability to visualize intact, developing organs is rapidly improving. Two-photon excitation allows less damaging penetration deeper into tissues with high resolution. Two-photon imaging and 3D reconstruction of the whole fetal mouse intestines in Walton et al., 2012 helped to define the pattern of villus outgrowth2. Here we describe a whole organ culture system that allows ex vivo development of villi and extensions of that culture system to allow the intestines to be three-dimensionally imaged during their development.

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Improved imaging technology is allowing three-dimensional imaging of organs during development. Here we describe a whole organ culture system that allows live imaging of the developing villi in the fetal mouse intestine.

Mouse fetale Whole Intestino Sistema Cultura per<em&gt; Ex Vivo</em&gt; La manipolazione delle vie di segnalazione e tridimensionale Imaging in diretta di Sviluppo del villo

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over ...

<meta charset="utf8"></head><body>Modello murino per lo studio dell'ileo postoperatorio attraverso la manipolazione intestinale

Postoperative Ileus Murine Model

Most patients experience postoperative ileus (POI) after surgery, which is associated with increased morbidity, mortality, and hospitalization time. POI is a consequence of mechanical damage during surgery, resulting in disruption of motility in the gastrointestinal tract. The mechanisms of POI are related to aberrant neuronal sensitivity, impaired epithelial barrier function, and increased local inflammation. However, the details remain enigmatic. Therefore, experimental murine models are crucial for elucidating the pathophysiology and mechanism of POI injury and for the development of novel therapies.
Here, we introduce a murine model of POI generated via intestinal manipulation (IM) that is similar to clinical surgery; this is achieved by mechanical damage to the small intestine by massaging the abdomen 1-3 times with a cotton swab. IM delayed gastrointestinal transit 24 h after surgery, as assessed by FITC-dextran gavage and fluorescence detection of the segmental digestive tract. Moreover, tissue swelling of the submucosa and immune cell infiltration were investigated by hematoxylin and eosin staining and flow cytometry. Proper pressure of the IM and a hyperemic effect on the intestine are critical for the procedure. This murine model of POI can be utilized to study the mechanisms of intestinal damage and recovery after abdominal surgery.

<meta charset="utf8"></head><body>Modello murino di ileo postoperatorio

We describe a murine model of postoperative ileus generated via intestinal manipulation. Gastrointestinal transit function, pathologic changes, and immune cell activation were assessed 24 h after surgery.

Modello murino di ileo postoperatorio

Most patients experience postoperative ileus (POI) after surgery, which is associated with increased morbidity, mortality, and hospitalization time. POI ...

Improved Swiss-rolling Technique for Intestinal Tissue Preparation for Immunohistochemical and Immunofluorescent Analyses

Understanding the role of factors that regulate intestinal epithelial homeostasis and response to injury and regeneration is important. The current literature describes several different methodological approaches to obtain images of intestinal tissues for data validation. In this paper, we delineate a common protocol relating to the derivation and processing of mouse intestinal tissues. Proper fixation of intestinal tissues and Swiss-roll techniques that enhance intestinal epithelial morphology are discussed. Postresection processing and reorientation of embedded intestinal tissues are critical in obtaining paraffin-embedded blocks that display intact intestinal structural features after sectioning. The Swiss-rolling technique helps in histological assessment of the complete intestinal or colonic sections examined. An ability to differentiate intestinal structural features can be vital in quantitative measurements of intestinal inflammation and tumorigenesis along the entire length. Finally, paraffin-embedded sections are ideal for robust processing using both immunohistochemical and immunofluorescent detection methods. Nonfluorescent immunohistochemical sections provide a vibrant image of the tissue detailing different cellular structural features but do not provide flexibility for intracellular co-localization experiments. Multiple fluorescent channels can be appropriately utilized with immunofluorescent detection for co-localization experiments, lending support to mechanistic studies.

Accurate identification and location of epithelial cells along the intestinal mucosal lining are essential to define different cell lineages. Proper imaging of intestinal tissues is crucial for identification of protein expression patterns with maximum resolution. This study aims to delineate the optimal methods and conditions for processing mouse intestinal tissues.

Miglioramento Tecnica Swiss-laminazione per intestinale Preparazione del tessuto per immunoistochimica e analisi immunofluorescenza

Understanding the role of factors that regulate intestinal epithelial homeostasis and response to injury and regeneration is important. The current ...

Un dispositivo semplice da preparare rapidamente intere Monti della Intestino mouse

Riepilogo

Esplora Altri Video

Protocolli per l'analisi del ruolo delle cellule Paneth in Rigenerazione della murino Intestino con condizionale

Valutazione funzionale della motilità intestinale e Wall infiammazione intestinale nei roditori: Analisi in un modello standardizzato di manipolazione intestinale

Immuno-fluorescenza etichettatura dei microtubuli e proteine centrosomali in tessuto intestinale Ex Vivo e 3D In Vitro intestinale Organoids

Il

Visualizzazione delle cellule interstiziali di Cajal (ICC) di rete in topi

Mouse fetale Whole Intestino Sistema Cultura per

Modello murino di ileo postoperatorio

Modello murino di ileo postoperatorio

Modello murino di ileo postoperatorio

Miglioramento Tecnica Swiss-laminazione per intestinale Preparazione del tessuto per immunoistochimica e analisi immunofluorescenza