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

연구는 농업과 아그리 - 푸드 캐나다 연구 센터, 레스 브리지, 앨버타, 캐나다의 헌신과 많은 사람의 노력없이 완료되지 않았을 수 있습니다. 그 점에 관해서는, 우리는 감사 : 사전 및 사후 수술 동물의 치료, 수술 준비 및 마취, 샘플의 분석 및 비디오 영상과 함께 도움을 타라 셸턴, 사전 및 사후 수술 치료에 대한 랜디 와일드와 에스텔라 코스타; 샘플 분석을 위해 제니 Gusse, 투관침 이미지 바이런 리 및 양의 축산을위한 브라이언 Egland. 우리는 또한 비디오 장비 및 조언뿐만 아니라 다른 도움을 (즉, 이그 제 큐 티브 생산) 제공 브렌트 셀린저을 (생물 과학학과, 레스 브리지 대학) 감사 및 교육 과정 다시 개발 센터, videography 전문 지식에 대한 레스 브리지 대학. 이 연구는 고급 식품 소재 네트워크 (GDI), 캐나다 앨버타 쇠고기 산업 개발 기금 (GDI), 그리고 농업과 아그리 - 식품 캐나다 (GDI와 JPK)에서 부여에 의해 일부 지원되었다.

실험 절차 : <ol><li> 모든 악기 autoclaved되며 이외 autoclaveable 자료는 10 분 최소 Germex 차가운 살균 용액에 소독을하고 있습니다. </li><li> 쉽 acepromazine (0.05 밀리그램 / kg), glycopyrrolate (0.005 밀리그램 / kg) 및 butorphanol tartrate의 (0.2 밀리그램 / kg) IM 사전 치료하고 있으며, 15-20 분 떠났다. </li><li> 목이 면도와 피부 수술 하찮은 것입니다. 경정맥 정맥 시각되면, lidocaine의 0.1 ML이 정맥을 통해 SC 주입, 카테터?...

<ol>
	<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>

이 절차에서는, 우리는 성공적으로 양 회장 내에 설립된 장 루프에 카테터를 설정할 수 있었다. 엄격한 사후 수술 치료 준수하면서, 동물은 빠르게 절차에서 발견한 및 수술 후 정상적인 활동 5-7일을 재개. 우리는 루프 내에서 카테터의 설립이 장의 점막이나 면역 기능 (데이터가 표시되지 않음)에 영향을주지 않고 동물들이 수술 후 최소한 8 주 살 수있는 결정. 이 절차는 우리가 한 동물 수술에서...

<table border="1"><tr><td> 카테고리 / 항목 번호 </td><td> 회사 </td><td> 카탈로그 </td></tr><tr><td colspan="3"> 외과 의사 공급 </td></tr><tr><td> 수술 장갑 </td><td> WDDC </td><td> 103091 </td></tr><tr><td> 외과 마스크 </td><td> WDDC </td><td> MAX53013 </td></tr><tr><td> 수술 가운 </td><td> CDMV B </td><td> 3365 </td></tr><tr><td> OB 에이프런 </td><td> WDDC </td><td> 900062 </td></tr><tr><td> 외과 캡 </td><td> WDDC </td><td> 903271 </td></tr><tr><td> EZ 스크럽 브러시 </td><td> WDDC </td><td> BDC377479 </td></tr><tr><td colspan="3"> 마취와 약물 </td></tr><tr><td> 마취 기계 </td><td> 보일 </td><td> </td></tr><tr><td> Sodasorb </td><td> WDDC </td><td> 108638 </td></tr><tr><td> 후두경 </td><td> WDDC </td><td> </td></tr><tr><td> 후두경 블레이드 (밀러 블레이드 # 4) </td><td> WDDC </td><td> 902667 </td></tr><tr><td> Endotracheal 튜브 (7.0-8.5 mm) </td><td> WDDC </td><td> </td></tr><tr><td> Lidocaine endotracheal 스프레이 </td><td> WDDC </td><td> 103364 </td></tr><tr><td> Acepromazine </td><td> WDDC </td><td> AUS01020 </td></tr><tr><td> Glycopyrrolate </td><td> WDDC </td><td> SAB2880 </td></tr><tr><td> Butorphanol tartrate의 </td><td> WDDC </td><td> AYE304541 </td></tr><tr><td> 다이아 제 팜 </td><td> WDDC </td><td> sab790 </td></tr><tr><td> Thiopental 나트륨 </td><td> WDDC </td><td> 103508 </td></tr><tr><td> 에피네프린 염산염 </td><td> WDDC </td><td> 103856 </td></tr><tr><td> Doxapram </td><td> 스트라 스 코나 약국 </td><td> </td></tr><tr><td> Isoflurane </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> 메트로니다졸 </td><td> WDDC </td><td> 101653 </td></tr><tr><td> 암피실린 </td><td> WDDC </td><td> NOV67185 - 6 </td></tr><tr><td> Cefazolin </td><td> WDDC </td><td> nov69397 </td></tr><tr><td colspan="3"> 수술 장비 및 소모품 </td></tr><tr><td> Electroscapel </td><td> Macan </td><td> MV8 </td></tr><tr><td> 난방 매트 </td><td> 칸 </td><td> PHMD - 48 </td></tr><tr><td> 위 튜브 (Nalgene 튜빙 16분의 5 &quot;ID) </td><td> 피셔 사이 언티픽 </td><td> </td></tr><tr><td> 니들 드라이버 </td><td> WDDC </td><td> 108338 </td></tr><tr><td> 애디슨 브라운 forcep </td><td> WDDC </td><td> 911075 </td></tr><tr><td> 랫 치아 forcep </td><td> CDMV </td><td> 5275 </td></tr><tr><td> 켈리 forcep </td><td> WDDC </td><td> 901133 </td></tr><tr><td> 모기 hemostat </td><td> WDDC </td><td> 108471 </td></tr><tr><td> Carmalt forcep </td><td> WDDC </td><td> 901225 </td></tr><tr><td> 메스 핸들 </td><td> WDDC </td><td> 900285 </td></tr><tr><td> 메스 블레이드 # 22 </td><td> WDDC </td><td> 900511 </td></tr><tr><td> Metzenbaum의 가위 </td><td> WDDC </td><td> 901166 </td></tr><tr><td> 날카로운 날카로운 가위 </td><td> WDDC </td><td> 108264 </td></tr><tr><td> 타월 클램프 </td><td> WDDC </td><td> 901213 </td></tr><tr><td> 앨리스 조직 forcep </td><td> WDDC </td><td> 901201 </td></tr><tr><td> 도옌의 forcep </td><td> WDDC </td><td> 904226 </td></tr><tr><td> 스테인리스 그릇 </td><td> WDDC </td><td> 903601 </td></tr><tr><td> 스테인리스 스틸 투관침 (곡선) </td><td> 지역 업체 </td><td> </td></tr><tr><td> 카테 테르의 introducer </td><td> WDDC </td><td> 11,440 </td></tr><tr><td> 외과 수건 </td><td> CDMV </td><td> 7972 </td></tr><tr><td> 일회용 드레이프 </td><td> WDDC </td><td> 900035 </td></tr><tr><td> Steri의 드레이프 </td><td> CDMV </td><td> 2323 </td></tr><tr><td> 드레이프 (40 X 40cm) </td><td> CDMV </td><td> 8358 </td></tr><tr><td> 외과 드레이프 (7 &quot;X 11&quot;오프닝) </td><td> CDMV </td><td> 269​​4 </td></tr><tr><td> Vicryl (2-0) </td><td> WDDC </td><td> JJMJ333H </td></tr><tr><td> 실크 2-0 </td><td> WDDC </td><td> 105248 </td></tr><tr><td> # 3 장선 </td><td> WDDC </td><td> CSL547 - 2070 </td></tr><tr><td> Supramid # 1 </td><td> WDDC </td><td> CSL547 - 3040 </td></tr><tr><td> 바늘 (절단 및 비 절삭) </td><td> WDDC </td><td> </td></tr><tr><td colspan="3"> 카테 테르 </td></tr><tr><td> Silastic 관 (0.040 &quot;ID, 0.085&quot;OD) </td><td> 피셔 사이 언티픽 </td><td> 11-189 - 15D </td></tr><tr><td> Orbeseal 연고 </td><td> WDDC </td><td> 106344 </td></tr><tr><td> 지우기 실리콘 (732 다목적 밀봉제) </td><td> 지역 업체 </td><td> </td></tr><tr><td> 블런트 최종 바늘 </td><td> WDDC </td><td> 905035 </td></tr><tr><td> 마커 (영구 산업) </td><td> 지역 업체 </td><td> </td></tr><tr><td> 카테 테르 파우치 (Elastoplast 7.5 cm의 건설) </td><td> WDDC </td><td> 104094 </td></tr><tr><td colspan="3"> 붕대 </td></tr><tr><td> 병원 테이프 (2 &quot;) </td><td> WDDC </td><td> 103842 </td></tr><tr><td> 병원 테이프 (1 &quot;) </td><td> WDDC </td><td> VLMRENF20 </td></tr><tr><td> 거즈 스폰지 4X4 </td><td> WDDC </td><td> 107551 </td></tr><tr><td> 거즈가 집착 </td><td> WDDC </td><td> 107633 </td></tr><tr><td> Elastoplast 7.5cm </td><td> WDDC </td><td> 104094 </td></tr><tr><td> Elastoplast rippable 붕대 </td><td> WDDC </td><td> 104093 </td></tr><tr><td colspan="3"> NEedles 및 주사기 </td></tr><tr><td> 주사기 (60 ML) </td><td> WDDC </td><td> 104004 </td></tr><tr><td> 주사기 (10 ML) </td><td> WDDC </td><td> bdc301604 </td></tr><tr><td> 25G 니들 </td><td> WDDC </td><td> BDC305122 </td></tr><tr><td> 20G 니들 </td><td> WDDC </td><td> BDC305175 </td></tr><tr><td> 18G 니들 </td><td> WDDC </td><td> 903353 </td></tr><tr><td colspan="3"> 정맥 </td></tr><tr><td> Intracath IV 카테터 (16G 8 &quot;) </td><td> WDDC </td><td> bdc3831621 </td></tr><tr><td> IV 관리 세트 (10 방울) </td><td> WDDC </td><td> 101797 </td></tr><tr><td> 0.9 % NaCl 용액 </td><td> WDDC </td><td> BAXJB1322P </td></tr><tr><td> Plasmalyte 148 솔루션 </td><td> WDDC </td><td> BAXJB2534 </td></tr><tr><td> 우선당 </td><td> WDDC </td><td> MCT60190 </td></tr><tr><td> 연동 펌프 </td><td> 콜 파머 </td><td> 7524-10 </td></tr><tr><td> 가축 식별 태그 시멘트 </td><td> 지역 업체 </td><td> </td></tr><tr><td colspan="3"> Sanatizers / disinfectants </td></tr><tr><td> Germex 솔루션 </td><td> WDDC </td><td> mtc60270 </td></tr><tr><td> 이소 프로필 알코올 </td><td> WDDC </td><td> 104590 </td></tr><tr><td> Stanhexidine의 클렌저 </td><td> WDDC </td><td> 109248 </td></tr><tr><td> Prepodyne 솔루션 </td><td> WDDC </td><td> 105710 </td></tr><tr><td> Prepodyne 스크럽 </td><td> WDDC </td><td> 105711 </td></tr></table> 서양 의약품 유통 센터 주식 회사, 에드먼턴, AB. B 베티 약물 유통 센터 주식 회사, 세인트 시아신쓰, QE.

catheterization of intestinal loops in ruminants

소장뿐만 아니라 영양분의 흡수에 관여하는 복잡한 구조이지만, 또한 개인과 외부 세계 사이의 장벽 역할을합니다. 따라서 소장은 immunosurveillance와 장용 병원균으로부터 보호에 중추적인 역할을합니다. 장 생리 및 면역학을 조사하는 것은 일반적으로 실험 모델로 &#39;창자 고리&#39;를 고용합니다. 이러한 루프 모델의 대부분은 소장에서 단기간 (&lt;24 시간) 변경 (1-3) 학업 이외의 회복 수술 절차입니다. 우리는 이전에 구체적으로 백신, adjuvants, 그리고 바이러스 (4) 양이 다음과 같은 노출의 소장의 장기 (&lt;6 MO) 면역 변화를 측정하기 위해 복구 장 루프 모델을 만들었습니다. 특정 &#39;루프&#39;이 절차는 현지화된 트리 트먼트, 우리가 소장의이 지역을 맛볼 수 있도록합니다. 이 방법의 큰 단점은 치료를 (수술 당시 IE) 관리 기회의 단일 창입니다. 또한 장의 점막과 luminal 내용을 모두 샘플은 프로젝트의 종료에서 찍은 수 있습니다. 위의 모델의 다른 분출 한계 수술 조작하고 필요한 사후 수술 조치 (항생제와 진통제의 예 기업청) 직접 치료 자체 및 /에 영향을 미치거나, 따라서 혼란함을 주죠 결과를 면역 기능을 변경할 수 있습니다. 따라서, 우리는 루프로 장기간 카테터를 삽입하여 장 루프 모델을 수정했습니다. 쉽 절차에서 완전히 회복하고 exteriorized 카테터에 의해 영향을받지 있습니다. 특히, 루프에서 카테터의 설립은 우리가 수술 중에 투여 약물의 수술과 통관의 복구 및 사후 수술 기간에 따라 연장 간격을 통해 여러 치료법을 소개하실 수 있습니다.

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

Catheterization of Intestinal Loops in Ruminants

우리는 양의 회장에서 &#39;창자 고리&#39;를 catheterizing위한 새로운 수술 방법을 설명합니다. 일단 동물이 수술에서 회복하고 항생제와 진통제를 통과해야 여러 치료는 카테터를 통해 루프에서 직접 입금하실 수 있습니다.

Ruminants에 창자 루프 Catheterization

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.

IBD의 DSS 유발 모델에서 창자 염증을 조사

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

연구

생물학

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.

차 소장 Organoid 문화에서 레트로 바이러스 감염의 비디오 프로토콜

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.

대사 프로파일의 실시간 분석<em&gt; 전의 VIVO</em&gt; 마우스 장내 크립트 Organoid 문화

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.

렌티 바이러스 형질 도입 및 장내 Organoids의 다운 스트림 분석을위한 프로토콜

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.

마우스에 압력 볼륨 루프 측정

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.

생체 외에서 그리고 Vivo에서 접근 장 상피 세포 침투성을 결정 하

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.

장 부분 방해의 마우스 모델

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.

이온화 조사에 대한 반응으로 장 상피 재생

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

확립된 쥐 대장에 대한 장 염증의 상피 효과

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.

저자 스포트라이트: 확립된 쥐 결장체에 대한 장 염증의 체외 상피 효과 연구  

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.

확립된 쥐 콜로노이드에 대한 시험관 내 장 염증의 상피 효과 연구

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