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

Het onderzoek kon niet worden voltooid zonder de inzet en inspanningen van vele personen aan de landbouw en Agri-Food Canada Research Centre, Lethbridge, Alberta, Canada. In dit verband, danken wij: Tara Shelton voor pre-en post-operatieve zorg van de dieren, chirurgische voorbereiding en anesthesiologie, analyse van de monsters, en hulp met video imaging; Randy Wilde en Estela Costa voor pre-en post-operatieve zorg; Jenny Gusse voor monsteranalyse, Byron Lee voor de trocar beeld, en Brian Egland voor schapen veeteelt. We danken ook Brent Selinger (Department of Biological Sciences, Universiteit van Lethbridge) voor het leveren van video-apparatuur en advies, alsmede andere bijstand (dat wil zeggen de uitvoerende productie), en het Curriculum Re-Development Centre, Universiteit van Lethbridge voor videografie expertise. Dit onderzoek werd mede ondersteund door subsidies van de Advanced Foods and Materials Network (GDI), het Canada-Alberta Beef Industry Development Fund (GDI), en landbouw en Agri-Food Canada (GDI en JPK).

Experimentele procedure: <ol><li> Alle instrumenten worden geautoclaveerd, niet-autoclaveerbaar materialen worden gesteriliseerd in Germex koude sterilisatie oplossing voor een minimum van 10 minuten. </li><li> Schapen zijn vooraf behandeld met acepromazine (0,05 mg / kg), glycopyrrolaat (0,005 mg / kg), en butorphanoltartraat (0,2 mg / kg) IM, en vertrok naar 15-20 minuten. </li><li> De hals is geschoren en de huid operatief verwijderd. Zodra de halsader is gevisualiseerd, 0,1 ml van lidocaïne wordt geïnjecteerd SC...

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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 deze procedure, konden we met succes katheters vestigen in de darm lussen opgericht in het ileum van schapen. Door vast te houden aan de strenge post-operatieve zorg, dieren zich snel hersteld van de procedure en de normale activiteiten hervat 5-7 dagen na de operatie. We hebben vastgesteld dat de oprichting van katheters in lussen niet intestinale mucosale of het immuunsysteem (gegevens niet getoond) beïnvloeden en dieren kunnen leven gedurende minstens 8 weken na de operatie. Deze procedure stelt ons in staat om m...

<table border="1"><tr><td> Categorie / Artikelnummer </td><td> Vennootschap </td><td> Catalogus </td></tr><tr><td colspan="3"> Chirurg levert </td></tr><tr><td> Chirurgische handschoenen </td><td> WDDC een </td><td> 103091 </td></tr><tr><td> Chirurgisch masker </td><td> WDDC </td><td> MAX53013 </td></tr><tr><td> Chirurgenjas </td><td> CDMV b </td><td> 3365 </td></tr><tr><td> OB Schort </td><td> WDDC </td><td> 900062 </td></tr><tr><td> Chirurgische cap </td><td> WDDC </td><td> 903271 </td></tr><tr><td> EZ Scrub borstel </td><td> WDDC </td><td> BDC377479 </td></tr><tr><td colspan="3"> Anesthesie en drugs </td></tr><tr><td> Verdoving machine </td><td> Boyle </td><td> </td></tr><tr><td> Sodasorb </td><td> WDDC </td><td> 108638 </td></tr><tr><td> Laryngoscoop </td><td> WDDC </td><td> </td></tr><tr><td> Laryngoscoop blad (Miller mes # 4) </td><td> WDDC </td><td> 902667 </td></tr><tr><td> Endotracheale tube (7,0 tot 8,5 mm) </td><td> WDDC </td><td> </td></tr><tr><td> Lidocaïne endotracheale spuiten </td><td> WDDC </td><td> 103364 </td></tr><tr><td> Acepromazine </td><td> WDDC </td><td> AUS01020 </td></tr><tr><td> Glycopyrrolaat </td><td> WDDC </td><td> SAB2880 </td></tr><tr><td> Butorphanoltartraat </td><td> WDDC </td><td> AYE304541 </td></tr><tr><td> Diazepam </td><td> WDDC </td><td> sab790 </td></tr><tr><td> Thiopentalnatrium </td><td> WDDC </td><td> 103508 </td></tr><tr><td> Epinefrine hydrochloride </td><td> WDDC </td><td> 103856 </td></tr><tr><td> Doxapram </td><td> Strathcona Apotheek </td><td> </td></tr><tr><td> Isofluraan </td><td> WDDC </td><td> 100339 </td></tr><tr><td> Hemostam </td><td> WDDC </td><td> 103857 </td></tr><tr><td> Flunixine </td><td> WDDC </td><td> sch140100 </td></tr><tr><td> Enrofloxacin </td><td> WDDC </td><td> hll0049 </td></tr><tr><td> Metronidazol </td><td> WDDC </td><td> 101653 </td></tr><tr><td> Ampicilline </td><td> WDDC </td><td> NOV67185-6 </td></tr><tr><td> Cefazoline </td><td> WDDC </td><td> nov69397 </td></tr><tr><td colspan="3"> Chirurgische apparatuur en benodigdheden </td></tr><tr><td> Electroscapel </td><td> Macan </td><td> MV8 </td></tr><tr><td> Verwarmingsmat </td><td> Kane </td><td> PHMD-48 </td></tr><tr><td> Maagsonde (Nalgene buis 5 / 16 &quot;ID) </td><td> Fisher Scientific </td><td> </td></tr><tr><td> Naald driver </td><td> WDDC </td><td> 108338 </td></tr><tr><td> Addison bruin pincet </td><td> WDDC </td><td> 911075 </td></tr><tr><td> Rat tand pincet </td><td> CDMV </td><td> 5275 </td></tr><tr><td> Kelly pincet </td><td> WDDC </td><td> 901133 </td></tr><tr><td> Mosquito hemostat </td><td> WDDC </td><td> 108471 </td></tr><tr><td> Carmalt pincet </td><td> WDDC </td><td> 901225 </td></tr><tr><td> Scalpel handvat </td><td> WDDC </td><td> 900285 </td></tr><tr><td> Scalpel # 22 </td><td> WDDC </td><td> 900511 </td></tr><tr><td> Metzenbaum schaar </td><td> WDDC </td><td> 901166 </td></tr><tr><td> Sharp-scherpe schaar </td><td> WDDC </td><td> 108264 </td></tr><tr><td> Handdoek klem </td><td> WDDC </td><td> 901213 </td></tr><tr><td> Allis weefsel pincet </td><td> WDDC </td><td> 901201 </td></tr><tr><td> Doyen&#39;s pincet </td><td> WDDC </td><td> 904226 </td></tr><tr><td> Roestvrijstalen kom </td><td> WDDC </td><td> 903601 </td></tr><tr><td> Roestvrij staal trocar (gebogen) </td><td> Lokale leverancier </td><td> </td></tr><tr><td> Catheter introducer </td><td> WDDC </td><td> 11440 </td></tr><tr><td> Chirurgische handdoek </td><td> CDMV </td><td> 7972 </td></tr><tr><td> Wegwerp draperen </td><td> WDDC </td><td> 900035 </td></tr><tr><td> Steri draperen </td><td> CDMV </td><td> 2323 </td></tr><tr><td> Laken (40 x 40 cm) </td><td> CDMV </td><td> 8358 </td></tr><tr><td> Chirurgische draperen (7 &quot;x 11&quot; opening) </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 # 3 </td><td> WDDC </td><td> CSL547-2070 </td></tr><tr><td> Supramid # 1 </td><td> WDDC </td><td> CSL547-3040 </td></tr><tr><td> Naalden (snijden en niet-snijdende) </td><td> WDDC </td><td> </td></tr><tr><td colspan="3"> Catheter </td></tr><tr><td> Silastic slang (0.040 &quot;ID, 0.085&quot; OD) </td><td> Fisher Scientific </td><td> 11 tot 189-15D </td></tr><tr><td> Orbeseal zalf </td><td> WDDC </td><td> 106344 </td></tr><tr><td> Helder silicone (732 multi-purpose kit) </td><td> Lokale leverancier </td><td> </td></tr><tr><td> Stompe einde naald </td><td> WDDC </td><td> 905035 </td></tr><tr><td> Marker (permanente industriële) </td><td> Lokale leverancier </td><td> </td></tr><tr><td> Katheter zakje (gemaakt van Hansaplast 7,5 cm) </td><td> WDDC </td><td> 104094 </td></tr><tr><td colspan="3"> Bandages </td></tr><tr><td> Ziekenhuis tape (2 &quot;) </td><td> WDDC </td><td> 103842 </td></tr><tr><td> Ziekenhuis tape (1 &quot;) </td><td> WDDC </td><td> VLMRENF20 </td></tr><tr><td> Gaas spons 4x4 </td><td> WDDC </td><td> 107551 </td></tr><tr><td> Gauze vastklampen </td><td> WDDC </td><td> 107633 </td></tr><tr><td> Hansaplast 7.5cm </td><td> WDDC </td><td> 104094 </td></tr><tr><td> Hansaplast rippable bandage </td><td> WDDC </td><td> 104093 </td></tr><tr><td colspan="3"> Needles en spuiten </td></tr><tr><td> Spuit (60 ml) </td><td> WDDC </td><td> 104004 </td></tr><tr><td> Spuit (10 ml) </td><td> WDDC </td><td> bdc301604 </td></tr><tr><td> 25G Naald </td><td> WDDC </td><td> BDC305122 </td></tr><tr><td> 20G Naald </td><td> WDDC </td><td> BDC305175 </td></tr><tr><td> 18G Naald </td><td> WDDC </td><td> 903353 </td></tr><tr><td colspan="3"> Intraveneus </td></tr><tr><td> Intracath IV katheter (16G 8 &quot;) </td><td> WDDC </td><td> bdc3831621 </td></tr><tr><td> Intraveneuze toediening (10 drop) </td><td> WDDC </td><td> 101797 </td></tr><tr><td> 0,9% NaCl-oplossing </td><td> WDDC </td><td> BAXJB1322P </td></tr><tr><td> Plasmalyte 148-oplossing </td><td> WDDC </td><td> BAXJB2534 </td></tr><tr><td> Dextrose </td><td> WDDC </td><td> MCT60190 </td></tr><tr><td> Slangenpomp </td><td> Cole Parmer </td><td> 7524-10 </td></tr><tr><td> Identificatie van vee tag cement </td><td> Lokale leverancier </td><td> </td></tr><tr><td colspan="3"> Sanatizers / ontsmettingsmiddelen </td></tr><tr><td> Germex oplossing </td><td> WDDC </td><td> mtc60270 </td></tr><tr><td> Isopropylalcohol </td><td> WDDC </td><td> 104590 </td></tr><tr><td> Stanhexidine reinigingsmiddel </td><td> WDDC </td><td> 109248 </td></tr><tr><td> Prepodyne oplossing </td><td> WDDC </td><td> 105710 </td></tr><tr><td> Prepodyne scrub </td><td> WDDC </td><td> 105711 </td></tr></table> een westerse Drug Distribution Center BV, Edmonton, AB. b De diergeneesmiddelen Distribution Center Inc, St. Hyacinthe, QE.

catheterization of intestinal loops in ruminants

De darm is een complexe structuur die is niet alleen betrokken in de opname van voedingsstoffen, maar fungeert ook als een barrière tussen het individu en de buitenwereld. Als zodanig, de darm speelt een centrale rol in immunosurveillance en bescherming tegen darmpathogenen. Onderzoek naar intestinale fysiologie en immunologie vaak heeft &#39;darm loops&#39; als een experimenteel model. De meerderheid van deze loop modellen zijn niet-inning chirurgische ingrepen die op korte termijn (&lt;24 uur) veranderingen in de darm (1-3) te bestuderen. We hebben eerder een herstel van darm-lus model om specifiek te meten op lange termijn (&lt;6 maanden) immunologische veranderingen in de darm van schapen na blootstelling aan vaccins, hulpstoffen, en virussen (4). Deze procedure gelokaliseerde behandelingen om een ​​specifieke &#39;lus&#39;, waardoor we dit deel van de darm monster. Een belangrijk nadeel van deze methode is de single window of opportunity om behandelingen (dwz op het moment van de operatie) te beheren. Bovendien kunnen monsters van zowel het darmslijmvlies en luminale inhoud alleen worden genomen bij de beëindiging van het project. Andere opvallende beperkingen van het bovenstaande model zijn dat de chirurgische manipulatie en noodzakelijke post-operatieve maatregelen (bijvoorbeeld de toediening van antibiotica en pijnstillers) direct kan de behandeling zelf en / beïnvloeden of wijzigen immuunsysteem, waardoor confounding resultaten. Daarom hebben we ons aangepast darm-lus model door het inbrengen van de lange termijn katheters in de lussen. Schapen volledig te herstellen van de procedure, en worden niet beïnvloed door de exteriorized katheters. Met name de vestiging van katheters in loops stelt ons in staat om meerdere behandelingen te introduceren over een langere periode, na herstel van een operatie en de klaring van geneesmiddelen toegediend tijdens de operatie en de postoperatieve periode.

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

Catheterization of Intestinal Loops in Ruminants

Beschrijven we een nieuwe chirurgische methode voor catheterizing &#39;darm loops&#39; in het ileum van schapen. Zodra de dieren hebben zich hersteld van een operatie en hebben opgeruimd antibiotica en pijnstillers, kunnen meerdere behandelingen direct worden gestort in lussen via de katheters.

Catheterisatie van de intestinale Loops bij herkauwers

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.

Onderzoeken darmontsteking in DSS-geïnduceerde Model van IBD

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

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.

Een video Protocol van retrovirale infectie in het jeugdwerk Intestinale organoïde Cultuur

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.

Real Time Analyse van Metabool profiel in<em&gt; Ex Vivo</em&gt; Muis Intestinale Crypt organoïde Cultures

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.

Een protocol voor Lentiviral Transduction en Downstream Analyse van Intestinale Organoids

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.

Het meten van druk Volume Loops in de muis

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 en In Vivo benaderingen te bepalen van intestinale epitheliale cel permeabiliteit

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.

Een muismodel van intestinale gedeeltelijke obstructie

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.

Intestinale epitheelregeneratie als reactie op ioniserende bestraling

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

Epitheliale effecten van darmontsteking op gevestigde muizencolonoïden

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.

Auteur in de schijnwerpers: onderzoek naar de epitheliale effecten van darmontsteking in vitro op gevestigde muizencolonoïden  

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.

Het bestuderen van de epitheliale effecten van darmontsteking in vitro op gevestigde murine colonoïden

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