Name: catheterization of intestinal loops in ruminants
Uploaded: 2009-06-11T00:00:00
Description: Watch this Scientific Journal Video about Catheterization of Intestinal Loops in Ruminants at JoVE.com

Die Forschung konnte nicht ohne das Engagement und die Bemühungen vieler Personen an der Agriculture and Agri-Food Canada Research Centre, Lethbridge, Alberta, Kanada abgeschlossen haben. In diesem Zusammenhang bedanken wir uns bei: Tara Shelton für Pre-und post-operative Betreuung von Tieren, OP-Vorbereitung und Anästhesie, Analyse von Proben und die Unterstützung mit Video-Bildgebung; Randy Wilde und Estela Costa für pre-und post-operative Versorgung; Jenny Gusse für die Probenanalyse, Byron Lee für den Trokar Bild, und Brian Egland für Schafhaltung. Wir danken auch Brent Selinger (Department of Biological Sciences, University of Lethbridge) für die Bereitstellung von Video-Ausrüstung und Beratung sowie sonstige Unterstützung (dh ausführende Produktion) und die Curriculum Re-Development Centre, University of Lethbridge für die Videografie Know-how. Diese Studie wurde zum Teil durch Zuschüsse aus dem erweiterten Foods und Materialien Network (GDI), das Kanada-Alberta Beef Industry Development Fund (GDI) und Agriculture and Agri-Food Canada (GDI und JPK) unterstützt.

Versuchsdurchführung: <ol><li> Alle Instrumente sind autoklaviert; nicht autoklavierbar Materialien sind in Germex Kaltsterilisation Lösung für ein Minimum von 10 min sterilisiert. </li><li> Schafe sind pre-medizinischen mit Acepromazin (0,05 mg / kg), Glycopyrrolat (0,005 mg / kg) und Butorphanoltartrat (0,2 mg / kg) IM, und links für 15-20 min. </li><li> Der Hals ist rasiert und die Haut chirurgisch abgetrennt. Sobald die Jugularvene wird visualisiert, 0,1 ml Lidocain injiziert wird SC über die Vene, den Kathet...

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	<li>Vidal, J. E., McClane, B. A., Saputo, J., Parker, J., Uzal, F. A. <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+Clostridium+perfringens+beta-toxin+on+the+rabbit+small+intestine+and+colon.">Effects of Clostridium perfringens beta-toxin on the rabbit small intestine and colon.</a> Infect. Immun. 76, 4396-4404 (2008).</li><li>Santos, R. L., Zhang, S., Tsolis, R. M., Baumler, A. J., Adams, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphologic+and+molecular+characterization+of+Salmonella+typhimurium+infection+in+neonatal+calves.">Morphologic and molecular characterization of Salmonella typhimurium infection in neonatal calves.</a> Vet. Pathol. 39, 200-215 (2002).</li><li>Uzzau, S., Leori, G. S., Petruzzi, V., Watson, P. R., Schianchi, G., Bacciu, D., Mazzarello, V., Wallis, T. S., Rubino, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salmonella+enterica+serovar-host+specificity+does+not+correlate+with+the+magnitude+of+intestinal+invasion+in+sheep.">Salmonella enterica serovar-host specificity does not correlate with the magnitude of intestinal invasion in sheep.</a> Infect. Immun. 69, 3092-3099 (2001).</li><li>Gerdts, V., Uwiera, R. R. E., Mutwiri, G. K., Wilson, D. J., Bowersock, T., Kindane, A., Babiuk, L. A., Griebel, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+intestinal+'loops'+provide+an+in+vivo+model+to+analyse+multiple+mucosal+immune+responses.">Multiple intestinal 'loops' provide an in vivo model to analyse multiple mucosal immune responses.</a> J. Immunol. Methods. 256, 19-33 (2001).</li><li>Greibel, P. J., Davis, W. C., Reynolds, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+analysis+of+the+growth+and+differentiation+of+b+cells+isolated+from+follicles+from+ileal+Peyer's+patch+of+sheep.">An analysis of the growth and differentiation of b cells isolated from follicles from ileal Peyer's patch of sheep.</a> Immunology. 75, 601-607 (1992).</li><li>Motyka, G., Greibel, P. J., Reynolds, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agents+that+activate+protein+kinase+C+resue+sheep+ileal+Peyer's+patch+B+cells+from+apoptosis.">Agents that activate protein kinase C resue sheep ileal Peyer's patch B cells from apoptosis.</a> Eur. J. Immunol. 23, 1314-1321 (1993).</li></ol>

In diesem Verfahren konnten wir erfolgreich etablieren Katheter in Darmschlingen im Ileum von Schafen hergestellt. Durch die Einhaltung strenger post-operative Betreuung, Tiere schnell wieder aus dem Verfahren und wieder normale Aktivität 5-7 Tage nach der Operation. Wir haben festgestellt, dass die Errichtung von Kathetern innerhalb von Schleifen hat keinen Einfluss auf Darmschleimhaut oder Funktion des Immunsystems (Daten nicht gezeigt) und Tiere können für mindestens 8 Wochen nach der Operation zu leben. Dieses Ve...

<table border="1"><tr><td> Kategorie / Artikelnummer </td><td> Firma </td><td> Katalog </td></tr><tr><td colspan="3"> Surgeon liefert </td></tr><tr><td> OP-Handschuhe </td><td> WDDC ein </td><td> 103091 </td></tr><tr><td> OP-Maske </td><td> WDDC </td><td> MAX53013 </td></tr><tr><td> OP-Kittel </td><td> CDMV b </td><td> 3365 </td></tr><tr><td> OB Schürze </td><td> WDDC </td><td> 900062 </td></tr><tr><td> Surgical cap </td><td> WDDC </td><td> 903271 </td></tr><tr><td> EZ Schrubber </td><td> WDDC </td><td> BDC377479 </td></tr><tr><td colspan="3"> Anästhesie und Medikamente </td></tr><tr><td> Narkosegerät </td><td> Boyle </td><td> </td></tr><tr><td> Sodasorb </td><td> WDDC </td><td> 108638 </td></tr><tr><td> Laryngoskop </td><td> WDDC </td><td> </td></tr><tr><td> Laryngoskopspatel (Miller Klinge # 4) </td><td> WDDC </td><td> 902667 </td></tr><tr><td> Endotrachealtubus (7,0 bis 8,5 mm) </td><td> WDDC </td><td> </td></tr><tr><td> Lidocain endotracheale Spray </td><td> WDDC </td><td> 103364 </td></tr><tr><td> Acepromazine </td><td> WDDC </td><td> AUS01020 </td></tr><tr><td> Glycopyrrolat </td><td> WDDC </td><td> SAB2880 </td></tr><tr><td> Butorphanoltartrat </td><td> WDDC </td><td> AYE304541 </td></tr><tr><td> Diazepam </td><td> WDDC </td><td> sab790 </td></tr><tr><td> Thiopental-Natrium </td><td> WDDC </td><td> 103508 </td></tr><tr><td> Adrenalin-Hydrochlorid </td><td> WDDC </td><td> 103856 </td></tr><tr><td> Doxapram </td><td> Strathcona Apotheke </td><td> </td></tr><tr><td> Isofluran </td><td> WDDC </td><td> 100339 </td></tr><tr><td> Hemostam </td><td> WDDC </td><td> 103857 </td></tr><tr><td> Flunixin </td><td> WDDC </td><td> sch140100 </td></tr><tr><td> Enrofloxacin </td><td> WDDC </td><td> hll0049 </td></tr><tr><td> Metronidazole </td><td> WDDC </td><td> 101653 </td></tr><tr><td> Ampicillin </td><td> WDDC </td><td> NOV67185-6 </td></tr><tr><td> Cefazolin </td><td> WDDC </td><td> nov69397 </td></tr><tr><td colspan="3"> Chirurgische Geräte und Zubehör </td></tr><tr><td> Electroscapel </td><td> Macan </td><td> MV8 </td></tr><tr><td> Heizmatte </td><td> Kane </td><td> PHMD-48 </td></tr><tr><td> Magensonde (Nalgene Schlauch 5 / 16 &quot;ID) </td><td> Fisher Scientific </td><td> </td></tr><tr><td> Needle-Treiber </td><td> WDDC </td><td> 108338 </td></tr><tr><td> Addison braun ForceP </td><td> WDDC </td><td> 911075 </td></tr><tr><td> Rat Zahn ForceP </td><td> CDMV </td><td> 5275 </td></tr><tr><td> Kelly ForceP </td><td> WDDC </td><td> 901133 </td></tr><tr><td> Mosquito hemostat </td><td> WDDC </td><td> 108471 </td></tr><tr><td> Carmalt ForceP </td><td> WDDC </td><td> 901225 </td></tr><tr><td> Skalpellgriff </td><td> WDDC </td><td> 900285 </td></tr><tr><td> Skalpell Nr. 22 </td><td> WDDC </td><td> 900511 </td></tr><tr><td> Metzenbaum Schere </td><td> WDDC </td><td> 901166 </td></tr><tr><td> Sharp scharfe Schere </td><td> WDDC </td><td> 108264 </td></tr><tr><td> Handtuch Klemme </td><td> WDDC </td><td> 901213 </td></tr><tr><td> Allis Gewebe ForceP </td><td> WDDC </td><td> 901201 </td></tr><tr><td> Doyen der ForceP </td><td> WDDC </td><td> 904226 </td></tr><tr><td> Schüssel aus Edelstahl </td><td> WDDC </td><td> 903601 </td></tr><tr><td> Edelstahl Trokar (gebogen) </td><td> Lokale Anbieter </td><td> </td></tr><tr><td> Katheter Einführhilfe </td><td> WDDC </td><td> 11440 </td></tr><tr><td> Chirurgische Handtuch </td><td> CDMV </td><td> 7972 </td></tr><tr><td> Einweg-Tuch </td><td> WDDC </td><td> 900035 </td></tr><tr><td> Steri drapieren </td><td> CDMV </td><td> 2323 </td></tr><tr><td> Drape (40 x 40 cm) </td><td> CDMV </td><td> 8358 </td></tr><tr><td> Chirurgische drapieren (7 &quot;x 11&quot; Öffnung) </td><td> CDMV </td><td> 2694 </td></tr><tr><td> Vicryl (2-0) </td><td> WDDC </td><td> JJMJ333H </td></tr><tr><td> Silk 2-0 </td><td> WDDC </td><td> 105248 </td></tr><tr><td> Catgut Nr. 3 </td><td> WDDC </td><td> CSL547-2070 </td></tr><tr><td> Supramid Nr. 1 </td><td> WDDC </td><td> CSL547-3040 </td></tr><tr><td> Needles (Schneide-und Nicht-Schneiden) </td><td> WDDC </td><td> </td></tr><tr><td colspan="3"> Katheter </td></tr><tr><td> Silastic Schlauch (0,040 &quot;ID, 0,085&quot; OD) </td><td> Fisher Scientific </td><td> 11 bis 189-15D </td></tr><tr><td> Orbeseal Salbe </td><td> WDDC </td><td> 106344 </td></tr><tr><td> Klare Silikon (732 Mehrzweck-Dichtmittel) </td><td> Lokale Anbieter </td><td> </td></tr><tr><td> Blunt Ende Nadel </td><td> WDDC </td><td> 905035 </td></tr><tr><td> Marker (permanent Industrie) </td><td> Lokale Anbieter </td><td> </td></tr><tr><td> Katheter Beutel (gebaut von Hansaplast 7,5 cm) </td><td> WDDC </td><td> 104094 </td></tr><tr><td colspan="3"> Bandagen </td></tr><tr><td> Krankenhaus-Band (2 &quot;) </td><td> WDDC </td><td> 103842 </td></tr><tr><td> Krankenhaus-Band (1 &quot;) </td><td> WDDC </td><td> VLMRENF20 </td></tr><tr><td> Gazetupfer 4x4 </td><td> WDDC </td><td> 107551 </td></tr><tr><td> Gauze klammern </td><td> WDDC </td><td> 107633 </td></tr><tr><td> Hansaplast 7,5 cm </td><td> WDDC </td><td> 104094 </td></tr><tr><td> Hansaplast rippable Verband </td><td> WDDC </td><td> 104093 </td></tr><tr><td colspan="3"> Needles und Spritzen </td></tr><tr><td> Spritze (60 ml) </td><td> WDDC </td><td> 104004 </td></tr><tr><td> Spritze (10 ml) </td><td> WDDC </td><td> bdc301604 </td></tr><tr><td> 25G Nadel </td><td> WDDC </td><td> BDC305122 </td></tr><tr><td> 20G Nadel </td><td> WDDC </td><td> BDC305175 </td></tr><tr><td> 18G Nadel </td><td> WDDC </td><td> 903353 </td></tr><tr><td colspan="3"> Intravenös </td></tr><tr><td> Intracath IV-Katheter (16G 8 &quot;) </td><td> WDDC </td><td> bdc3831621 </td></tr><tr><td> IV-Administration (10 Tropfen) </td><td> WDDC </td><td> 101797 </td></tr><tr><td> 0,9% NaCl-Lösung </td><td> WDDC </td><td> BAXJB1322P </td></tr><tr><td> Plasmalyte 148 Lösung </td><td> WDDC </td><td> BAXJB2534 </td></tr><tr><td> Traubenzucker </td><td> WDDC </td><td> MCT60190 </td></tr><tr><td> Schlauchpumpe </td><td> Cole Parmer </td><td> 7524-10 </td></tr><tr><td> Livestock Kennzeichnungsanhänger Zement </td><td> Lokale Anbieter </td><td> </td></tr><tr><td colspan="3"> Sanatizers / Desinfektionsmittel </td></tr><tr><td> Germex Lösung </td><td> WDDC </td><td> mtc60270 </td></tr><tr><td> Isopropylalkohol </td><td> WDDC </td><td> 104590 </td></tr><tr><td> Stanhexidine Reiniger </td><td> WDDC </td><td> 109248 </td></tr><tr><td> Prepodyne Lösung </td><td> WDDC </td><td> 105710 </td></tr><tr><td> Prepodyne scrub </td><td> WDDC </td><td> 105711 </td></tr></table> eine westliche Drug Distribution Center LTD, Edmonton, AB. b Die Veterinärpräparate Distribution Center Inc., St. Hyacinthe, QE.

catheterization of intestinal loops in ruminants

Der Darm ist ein komplexes Gebilde, das nicht nur in die Aufnahme von Nährstoffen beteiligt ist, sondern fungiert auch als eine Barriere zwischen dem Individuum und der Außenwelt. Insofern spielt der Darm eine zentrale Rolle in Immunüberwachung und Schutz vor Darm-Erreger. Untersuchung Darm-Physiologie und Immunologie allgemein beschäftigt &quot;Darmschlingen&quot; als ein experimentelles Modell. Die Mehrzahl dieser Schleife Modelle sind Nichtwiedereinziehung operative Eingriffe, die kurzfristig (&lt;24 h) Veränderungen im Darm (1-3) zu studieren. Wir zuvor erstellte eine Erholung Darmschlinge Modell gezielt messen langfristige (&lt;6 Monate) immunologische Veränderungen im Darm der Schafe nach Exposition mit Impfstoffen, Adjuvantien und Viren (4). Dieses Verfahren lokalisiert auf einen speziellen &#39;Schleife&#39;, so dass wir diesen Bereich des Darms Probe. Ein wesentlicher Nachteil dieser Methode ist das einzige Fenster der Gelegenheit, Behandlungen (dh zum Zeitpunkt der Operation) zu verwalten. Darüber hinaus können Proben sowohl der Darmschleimhaut und luminalen Inhalte nur bei Beendigung des Projekts getroffen werden. Sonstige wichtige Einschränkungen der oben genannten Modell, dass die chirurgische Manipulation und erforderliche postoperative Maßnahmen (zB Verabreichung von Antibiotika und Analgetika) kann direkt Einfluss auf die Behandlung selbst und / oder zu verändern Immunfunktion, damit verwirrende Ergebnisse. Deshalb haben wir unsere Darmschlinge Modell, indem Sie langfristige Katheter in die Schlaufen geändert. Sheep vollständig erholen von dem Verfahren und sind unabhängig von den exteriorisierten Kathetern. Insbesondere ermöglicht die Einrichtung von Kathetern in Schlaufen uns mehrere Behandlungen über einen längeren Zeitraum einführen, nach Erholung von der Operation und die Clearance von Medikamenten während der Operation verabreicht und die postoperativen Phase.

Watch this Scientific Journal Video about Catheterization of Intestinal Loops in Ruminants at JoVE.com

Catheterization of Intestinal Loops in Ruminants

Wir beschreiben eine neuartige OP-Methode zur Katheterisierung &quot;Darmschlingen&quot; im Ileum von Schafen. Sobald Tiere haben sich von der Operation erholt haben und gelöscht Antibiotika und Analgetika können mehrere Behandlungen direkt in Schlaufen über den Katheter hinterlegt.

Katheterisierung der Darmschlingen bei Wiederkäuern

The intestine is a complex structure that is involved not only in absorption of nutrients, but also acts as a barrier between the individual and the ...

catheterization-of-intestinal-loops-in-ruminants

Research

JoVE Journal

Biology

Investigating Intestinal Inflammation in DSS-induced Model of IBD

Inflammatory bowel disease (IBD) encompasses a range of intestinal pathologies, the most common of which are ulcerative colitis (UC) and Crohn's Disease (CD). Both UC and CD, when present in the colon, generate a similar symptom profile which can include diarrhea, rectal bleeding, abdominal pain, and weight loss.1 Although the pathogenesis of IBD remains unknown, it is described as a multifactorial disease that involves both genetic and environmental components.2
There are numerous and variable animal models of colonic inflammation that resemble several features of IBD. Animal models of colitis range from those arising spontaneously in susceptible strains of certain species to those requiring administration of specific concentrations of colitis-inducing chemicals, such as dextran sulphate sodium (DSS). Chemical-induced models of gut inflammation are the most commonly used and best described models of IBD. Administration of DSS in drinking water produces acute or chronic colitis depending on the administration protocol.3 Animals given DSS exhibit weight loss and signs of loose stool or diarrhea, sometimes with evidence of rectal bleeding.4,5 Here, we describe the methods by which colitis development and the resulting inflammatory response can be characterized following administration of DSS. These methods include histological analysis of hematoxylin/eosin stained colon sections, measurement of pro-inflammatory cytokines, and determination of myeloperoxidase (MPO) activity, which can be used as a surrogate marker of inflammation.6
The extent of the inflammatory response in disease state can be assessed by the presence of clinical symptoms or by alteration in histology in mucosal tissue. Colonic histological damage is assessed by using a scoring system that considers loss of crypt architecture, inflammatory cell infiltration, muscle thickening, goblet cell depletion, and crypt abscess.7 Quantitatively, levels of pro-inflammatory cytokines with acute inflammatory properties, such as interleukin (IL)-1&beta;, IL-6 and tumour necrosis factor (TNF)-&alpha;,can be determined using conventional ELISA methods. In addition, MPO activity can be measured using a colorimetric assay and used as an index of inflammation.8
In experimental colitis, disease severity is often correlated with an increase in MPO activity and higher levels of pro-inflammatory cytokines. Colitis severity and inflammation-associated damage can be assessed by examining stool consistency and bleeding, in addition to assessing the histopathological state of the intestine using hematoxylin/eosin stained colonic tissue sections. Colonic tissue fragments can be used to determine MPO activity and cytokine production. Taken together, these measures can be used to evaluate the intestinal inflammatory response in animal models of experimental colitis.

Experimental models of inflammatory bowel disease have allowed us to examine the complex innate and adaptive immune responses associated with pathogenesis. Using histological scoring, quantification of pro-inflammatory cytokines and myeloperoxidase activity, one can begin to assess these responses seen in inflammatory bowel disease.

Untersuchung Darmentzündungen in DSS-induzierten Modell der IBD

Inflammatory bowel disease (IBD) encompasses a range of intestinal pathologies, the most common of which are ulcerative colitis (UC) and Crohn's Disease ...

Forschung

Biologie

A Video Protocol of Retroviral Infection in Primary Intestinal Organoid Culture

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding primary 3D epithelial structures in vitro. Both the architecture and physiological properties of these &#39;mini-guts&#39;, also called organoids, closely resemble their in vivo counterparts. This makes them an attractive model system for the small intestinal epithelium. Using retroviral transduction, functional genetics can now be performed by conditional gene overexpression or knockdown. This video demonstrates the procedure of organoid culture, the generation of retroviruses, and the retroviral transduction of organoids to assist phenotypic analysis of the small intestinal epithelium in vitro. This novel organotypic model system in combination with retroviral mediated gene expression provides a valuable tool for rapid analysis of gene function in vitro without the need of costly and time-consuming generation for transgenic animals.

This protocol explains primary Lgr5-positve organoid culture and the subsequent performance of retroviral transduction. This enables Cre-inducible overexpression or knockdown of the delivered transgene and allows functional studies to be carried out in the novel in vitro organotypic model system.

Ein Video-Protokoll von retrovirale Infektion in der Grunddarm Organoide Kultur

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding ...

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and composed of mature enterocytes, goblet cells and enteroendocrine cells; and crypts, residing proximal to the submucosa and the muscularis, harboring adult stem and progenitor cells and mature Paneth cells, as well as stromal and immune cells of the crypt microenvironment. Until the last few years, in vitro studies of small intestine was limited to cell lines derived from either benign or malignant tumors, and did not represent the physiology of normal intestinal epithelia and the influence of the microenvironment in which they reside. Here, we demonstrate a method adapted from Sato et al. (2009) for culturing primary mouse intestinal crypt organoids derived from C57BL/6 mice. In addition, we present the use of crypt organoid cultures to assay the crypt metabolic profile in real time by measurement of basal oxygen consumption, glycolytic rate, ATP production and respiratory capacity. Organoids maintain properties defined by their source and retain aspects of their metabolic adaptation reflected by oxygen consumption and extracellular acidification rates. Real time metabolic studies in this crypt organoid culture system are a powerful tool to study crypt organoid energy metabolism, and how it can be modulated by nutritional and pharmacological factors.

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

Small intestinal crypt organoids cultured ex vivo provide a tissue culture system that recapitulates growth of crypts dependent on stem cells and their niche. We established a method to assay the metabolic profile in real time in primary mouse crypt organoids. We found organoids maintain physiological properties defined by their source.

Echtzeit-Analyse von Stoffwechselprofil in<em&gt; Ex Vivo</em&gt; Maus Intestinale Crypt Organoide Kulturen

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and ...

A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth factors. Since organoids are highly similar to the original tissue in terms of homeostatic stem cell differentiation, cell polarity and presence of all terminally differentiated cell types known to the adult intestinal epithelium, they serve as an essential resource in experimental research on the epithelium. The possibility to express transgenes or interfering RNA using lentiviral or retroviral vectors in organoids has increased opportunities for functional analysis of the intestinal epithelium and intestinal stem cells, surpassing traditional mouse transgenics in speed and cost. In the current video protocol we show how to utilize transduction of small intestinal organoids with lentiviral vectors illustrated by use of doxycylin inducible transgenes, or IPTG inducible short hairpin RNA for overexpression or gene knockdown. Furthermore, considering organoid culture yields minute cell counts that may even be reduced by experimental treatment, we explain how to process organoids for downstream analysis aimed at quantitative RT-PCR, RNA-microarray and immunohistochemistry. Techniques that enable transgene expression and gene knock down in intestinal organoids contribute to the research potential that these intestinal epithelial structures hold, establishing organoid culture as a new standard in cell culture.

In this video protocol we give a step by step explanation of lentiviral transduction in organoids of primary intestinal epithelium and of processing and downstream analysis of these cultures by quantitative RT-PCR, RNA-microarray and immunohistochemistry.

Ein Protokoll für Lentivirale Transduktion und Downstream-Analyse der Darm Organoide

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth ...

Measuring Pressure Volume Loops in the Mouse

Understanding the causes and progression of heart disease presents a significant challenge to the biomedical community. The genetic flexibility of the mouse provides great potential to explore cardiac function at the molecular level. The mouse&#39;s small size does present some challenges in regards to performing detailed cardiac phenotyping. Miniaturization and other advancements in technology have made many methods of cardiac assessment possible in the mouse. Of these, the simultaneous collection of pressure and volume data provides a detailed picture of cardiac function that is not available through any other modality. Here a detailed procedure for the collection of pressure-volume loop data is described. Included is a discussion of the principles underlying the measurements and the potential sources of error. Anesthetic management and surgical approaches are discussed in great detail as they are both critical to obtaining high quality hemodynamic measurements. The principles of hemodynamic protocol development and relevant aspects of data analysis are also addressed.

This manuscript describes a detailed protocol for the collection of pressure-volume data from the mouse.

Mess Druck Volumen Loops in der Maus

Understanding the causes and progression of heart disease presents a significant challenge to the biomedical community. The genetic flexibility of the ...

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and epithelial cell integrity, and disruption of the intestinal barrier function contributes to progression of gastrointestinal and systemic disease. Two simple methods are described here to measure the permeability of intestinal epithelium. In vitro, Caco-2BBe cells are plated in tissue culture wells as a monolayer and transepithelial electrical resistance (TER) can be measured by an epithelial (volt/ohm) meter. This method is convincing because of its user-friendly operation and repeatability. In vivo, mice are gavaged with 4 kDa fluorescein isothiocyanate (FITC)-dextran, and the FITC-dextran concentrations are measured in collected serum samples from mice to determine the epithelial permeability. Oral gavage provides an accurate dose, and therefore is the preferred method to measure the intestinal permeability in vivo. Taken together, these two methods can measure the permeability of the intestinal epithelium in vitro and in vivo, and hence be used to study the connection between diseases and barrier function.

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

Two methods are presented here to determine intestinal barrier function. An epithelial meter (volt/ohm) is used for measurements of transepithelial electrical resistance of cultured epithelia directly in tissue culture wells. In mice, the FITC-dextran gavage method is used to determine the intestinal permeability in vivo.

In Vitro und In Vivo Ansätze Darm Epithelzelle Durchlässigkeit zu bestimmen

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and ...

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.

Ein Maus-Modell der teilweisen Darmverschluss

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

Intestinal Epithelial Regeneration in Response to Ionizing Irradiation

The intestinal epithelium consists of a single layer of cells yet contains multiple types of terminally differentiated cells, which are generated by the active proliferation of intestinal stem cells located at the bottom of intestinal crypts. However, during events of acute intestinal injury, these active intestinal stem cells undergo cell death. Gamma irradiation is a widely used colorectal cancer treatment, which, while therapeutically efficacious, has the side effect of depleting the active stem cell pool. Indeed, patients frequently experience gastrointestinal radiation syndrome while undergoing radiotherapy, in part due to active stem cell depletion. The loss of active intestinal stem cells in intestinal crypts activates a pool of typically quiescent reserve intestinal stem cells and induces dedifferentiation of secretory and enterocyte precursor cells. If not for these cells, the intestinal epithelium would lack the ability to recover from radiotherapy and other such major tissue insults. New advances in lineage-tracing technologies allow tracking of the activation, differentiation, and migration of cells during regeneration and have been successfully employed for studying this in the gut. This study aims to depict a method for the analysis of cells within the mouse intestinal epithelium following radiation injury.

The gastrointestinal tract is one of the most sensitive organs to injury upon radiotherapeutic cancer treatments. It is simultaneously an organ system with one of the highest regenerative capacities following such insults. The presented protocol describes an efficient method to study the regenerative capacity of the intestinal epithelium.

Regeneration des Darmepithels als Reaktion auf ionisierende Bestrahlung

The intestinal epithelium consists of a single layer of cells yet contains multiple types of terminally differentiated cells, which are generated by the ...

Epithelale Auswirkungen von Darmentzündungen auf etablierte murine Kolonoide

Studying the Epithelial Effects of Intestinal Inflammation In Vitro on Established Murine Colonoids

The intestinal epithelium plays an essential role in human health, providing a barrier between the host and the external environment. This highly dynamic cell layer provides the first line of defense between microbial and immune populations and helps to modulate the intestinal immune response. Disruption of the epithelial barrier is a hallmark of inflammatory bowel disease (IBD) and is of interest for therapeutic targeting. The 3-dimensional colonoid culture system is an extremely useful in vitro model for studying intestinal stem cell dynamics and epithelial cell physiology in IBD pathogenesis. Ideally, establishing colonoids from the inflamed epithelial tissue of animals would be most beneficial in assessing the genetic and molecular influences on disease. However, we have shown that in vivo epithelial changes are not necessarily retained in colonoids established from mice with acute inflammation. To address this limitation, we have developed a protocol to treat colonoids with a cocktail of inflammatory mediators that are typically elevated during IBD. While this system can be applied ubiquitously to various culture conditions, this protocol emphasizes treatment on both differentiated colonoids and 2-dimensional monolayers derived from established colonoids. In a traditional culture setting, colonoids are enriched with intestinal stem cells, providing an ideal environment to study the stem cell niche. However, this system does not allow for an analysis of the features of intestinal physiology, such as barrier function. Further, traditional colonoids do not offer the opportunity to study the cellular response of terminally differentiated epithelial cells to proinflammatory stimuli. The methods presented here provide an alternative experimental framework to address these limitations. The 2-dimensional monolayer culture system also offers an opportunity for therapeutic drug screening ex vivo. This polarized layer of cells can be treated with inflammatory mediators on the basal side of the cell and concomitantly with putative therapeutics apically to determine their utility in IBD treatment.

Autor im Rampenlicht: Untersuchung der epithelialen Auswirkungen von Darmentzündungen in vitro auf etablierte murine Kolonoide  

We describe a protocol detailing the isolation of murine colonic crypts for the development of 3-dimensional colonoids. The established colonoids can then be terminally differentiated to reflect the cellular composition of the host epithelium prior to receiving an inflammatory challenge or being directed to establish an epithelial monolayer.

Untersuchung der epithelialen Effekte von intestinalen Entzündungen in vitro auf etablierte murine Kolonoide

The intestinal epithelium plays an essential role in human health, providing a barrier between the host and the external environment. This highly dynamic ...