Name: ex vivo intestinal sacs to assess mucosal permeability in models of gastrointestinal disease
Uploaded: 2016-02-09T13:00:00.000+00:00
Duration: 6 min 4 s
Description: Watch this Scientific Journal Video about Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease at JoVE.com

This work was funded by National Health and Medical Research Project Grant APP1021582 and a Hunter Medical Research Institute grant sponsored by Sparke Helmore/NBN Triathlon and the Estate of the late Leslie Kenneth McFarlane.

이 프로토콜의 모든 동물의 작업 절차를 승인 뉴캐슬 동물 윤리위원회의 대학 엄격하게 준수하여 수행됩니다. 악기, 문화 미디어와 요리 1. 준비 <ol><li> 37 미리 따뜻한 미디어 199 (TC199) 또는 둘 베코의 수정 독수리 중간 (DMEM)는 미디어 기음. 사전 네이트 95 %, O2 / 5 % CO 2로 버블 링하여 매체. 매체가 7.3의 최종 pH가 있는지 확인합니다. </li><li> 각 주머니 두 5cm 섹션을 절단하여 봉합을 준비합니다. 루프 닫히지 않은 매듭에 봉합. </li></ol> 2. 해부 및 위장관의 제조 <ol><li> 안락사 전에 고체 음식을 12 시간 인출. 원하는 경우,이 기간 동안 영양 젤 보충에 동물을 배치합니다. </li><li> 나트륨 펜토 바르비 과다 복용에 의한 쥐를 안락사 ([200 ㎎ / ㎏, 복강 내 주사) 기관 윤리 프로토에 따라 자궁 경부 전위 다음COLS는 복부와 흉부에 70 % 에탄올을 스프레이. </li><li> 가위 사용하여 복부의 중앙에 수평 절개를하고 복막을 노출. </li><li> 분리 유문 괄약근의 위의 상부 소장을 절단하고 항문에서 대장을 절단하여 위장관을 제거하기 위해 진행합니다. 부드럽게 장간막을 제거하기 위해 집게를 사용합니다. 미리 예열, 산소 매체의 창자를 놓습니다. </li><li> 소장의 부분을 식별하는 투과성 (그림 1)에 대한 평가 및 장관의 나머지에서 무료로이 부분을 절단한다. <ol><li> 동물 사이의 일관성을 유지 위장, 십이지장 및 소장의 상대적인 부분을 측정하고, 맹장 및 결장 회장 상대적인 부분을 측정하기 위해. </li><li> 조직 세그먼트를 선택하는 경우, 이러한 패치 파이어 등 점막 - 관련 림프 조직의 존재를 참고. 이러한 작은 끄덕임으로 식별 될 수있다루멘의 장막 측 ules. </li><li> 1 ML의 주사기를 사용하여 부드럽게 예열 PBS (37 ° C)와 페트리 접시에 장 세그먼트의 내강 내용을 세척하십시오. 이 대변 내용에 폐기되거나 저장 될 수 -80 원하는대로 향후 분석을 위해 C를 °. </li></ol></li></ol> 장내 주머 니나 3. 준비 <ol><li> 시험 화합물 또는 분자의 300 μL 부피 1 mL를 주사기를 준비한다. 점막의 무결성, FITC - 덱스 트란 M.Wt.의 1 ㎎ / ㎖ 솔루션 4400이 사용될 수있다. 4,400-70,000 다에서 크기에 이르기까지 프로브가 증가 감도를 사용할 수있다. 안전하게 주사기에 작은 동물 혈관 카테터를 장착한다. </li><li> 장 세그먼트의 오프닝에서 5cm 측정 및 세그먼트를 묶어 안전하게이 시점에서 봉합 루프로 마감했다. 조심스럽게 소장의 개구부 주위에 미리 연결 봉합 루프를 배치하고 무딘 카테터를 삽입합니다. 장 세그먼트를 확보 할 수 있도록 폐쇄 올가미를 당겨모든 용액을 주입 보장, 소장에 주사기에서 300 μl의 볼륨을 놓습니다. </li><li> 동시에 장 낭의 폐쇄를 고정 봉합 올가미를 잡아 당기면서 부드럽게 카테터를 제거합니다. 소장에서 느슨한 장 주머니를 잘라 37로 예열 산소 매체의 20 ㎖로 가득 50 ML 원뿔 관에 배치 기음. </li></ol> 투수 4. 측정 <ol><li> 가열 된 물을 욕조에 장 주머니를 포함 장소 원뿔 튜브는 37로 설정 기음. 0, 30, 60, 90 및 120 분의 시점에서, 각각의 인스턴스에 신선한 매체와 함께 100 ㎕의 부피를 대체하는 96 웰 플레이트 원뿔 튜브 및 전송에서 100 μL 샘플을 취할. </li><li> 최종 샘플을 취한 후, 점막 표면을 노출시키는, 봉합사 시점과 세그먼트의 길이 아래로 열린 주머니를 잘랐다. </li><li> 각 장 세그먼트의 길이 및 폭을 측정한다. 다목적 경우빨강, 스냅 -80에서 세그먼트 저장을 동결 분자 분석을위한 RNA 안정화 용액에서 단백질 또는 생화학 적 분석, 또는 대안 적으로, 상점에 대한 C를 °. </li><li> 1 1 × 10 -6까지 FITC-태그 분자 로그 희석의 표준 곡선을 구축합니다. </li><li> 형광 플레이트 리더에 FITC에 대한 측정 시료와 표준, FITC 여기 / 방출 / 519 내지 495 나노 미터. </li></ol> 각 개인 소장 골목에 대한 명백한 투수 5. 계산 <ol><li> 초에 시간 단위로 변환합니다. </li><li> 각 타임 포인트에 대한 누적 농도, Q를 계산할 Q의 t = (C t의 *의 V r에) + (Q t 합 * V s의), 장소 : Q의 t = 시간 t에서 누적 농도 C의 t = 시간 t에서의 농도 수신 측에서 V의 R = 볼륨 이전의 모든 Q의 t의 Q T는 합계 = 합계 V S =볼륨 샘플링 </li><li> 플롯 시간 (T) 대 Q 및 기울기를 계산 : δQ / ΔT </li><li> 명백한 투과성을 계산 (P 응용 프로그램) P 앱 = (δQ / ΔT) / (A * 공동), 장소 : 조직의 면적 A = C 0 = 초기 농도 </li></ol>

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저자는 더 경쟁 재정적 이해 관계가 없음을 선언합니다.

여기서는 점막 장벽 기능 생체을 평가 단리 장내 주머니의 제조 상세한있다. 소장 SAC 제제는 주로 소장 전체 후보 약물의 흡수를 시험하는 제약 연구에서 사용되어왔다. 그러나 이러한 분석은 동일하게 장 질환의 연구를 위해 적합하다. 지역 및 투과성의 사이트에 특정 평가에 따라 크게 다를 수 있습니다 장 투과성은 소화 질환 점막의 무결성의 지역 중요성에 대한 이해를 할 수 있습니다....

위장관 장 상피 장벽 인간 성인 400 평방 미터로 추정 점막 표면적이다. 따라서, 지속적 미생물 섭취 약물, 영양분 및 박테리아 독소의 도전에 노출된다. 호스트는 허용 공생 박테리아 병원균 구분하지해야하지만 동시에 영양소 흡수를 허용하면서, 상피 장벽을 넘어에서 이러한 종의 분자 및 분비를 방지한다. 따라서, 장 상피 세포의 역할은 내강 내용 1 선택적 장벽으로 작용한다. 이 구성 적 및 유도 메커니즘이 이루어진 반응 시스템을 통해 생물학적 작용 점막 상피에서 타고난 방어 시스템에 의해 부분적으로 달성된다. 상피 장벽 기능의 상실은 위장관 질환의 특징 인 다수의 병리이다. 생체를상피 장벽 기능의 검사는 추적 분자의 구강 투여 이후 혈청 분석 (3)를 통해 평가 될 수있다. 그러나,이 기술은 장벽 기능 장애의 사이트에 같은 징후를 제공하지 않습니다. 생체 외 및 트랜스 웰 시스템 (3)를 사용하여 각각 챔버 4,5 Ussing transepithelial 저항의 생체 평가에서 일반적으로 상피 장벽 기능의 대리 지표로 사용하지만, 동물 모델 (6)의 기여 질병 생리학 부족합니다. 이 프로토콜에서는 다수의 레벨에서 점막 장벽 기능을 평가하는데 사용될 수있다 장 무결성 및 직접 지역화 평가 수 생체 조직 준비 모델을 설명한다. 중요한 것은,이 방법은 질환의 동물 모델에 적용 할 수 있거나, 또는 그의 약학 적 점막 방벽 기능 부전의 깊이 질의에서 허용하도록 조작 될 수있다.

<table><tbody><tr><td>Dekantel&amp;nbsp; Non-absorbable Silk suture</td><td>Braintree Scientific</td><td>SUT-S 116</td><td></td></tr><tr><td>Media 199 (TC199)&amp;nbsp;</td><td>Life Technologies</td><td>11043-023</td><td>No phenol red as this interferes with fluorescence</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>Life Technologies</td><td>21063-045</td><td>No phenol red as this interferes with fluorescence</td></tr><tr><td>N-acetylcysteine</td><td>Sigma Aldrich</td><td></td><td>Use at 10 mM in media</td></tr><tr><td>Small animal vascular cathether: Physiocath</td><td>Data Sciences International</td><td>277-1-002</td><td></td></tr><tr><td>FITC-Dextran 4,400 MW</td><td>Sigma Aldrich</td><td>FD-4</td><td></td></tr><tr><td>FITC-Dextran 20,000 MW</td><td>Sigma Aldrich</td><td>FD-20</td><td></td></tr><tr><td>FITC-Dextran 70,000 MW</td><td>Sigma Aldrich</td><td>FD-70</td><td></td></tr></tbody></table>

ex vivo intestinal sacs to assess mucosal permeability in models of gastrointestinal disease

상피 장벽 위장관 제 타고난 방어 선택적 작은 상피 걸쳐 분자와 거의 상피 고분자 전송 금지의 이동을 제한하는 하부 조직 구획 루멘으로부터 수송을 조절한다. 이 선택은 친 유성 분자 양쪽 정점 수용체 상피 단단한 접합부 단백질 복합체의 전송을 제한 점액 겔 층에 의해 결정된다. 상피 세포의 시험 관내 세포 배양 모델은 편리하지만, 모델, 그들은 부족한 미생물, 점액 젤, 상피 세포와 면역 체계 사이의 상호 작용의 복잡성. 한편, 사이트 특이성 표시없이 장내 흡수 또는 투과율의 생체 내 평가를 수행 할 수 있지만, 이러한 분석은 전체 위장관 흡수를 측정한다. 생체 외 투과 검정을 &quot;장 주머니&quot;를 사용하여; 장 사이트 특이성의 추가적인 장점으로, 전체 장 무결성 또는 특정 분자의 비교 수송를 측정하는 신속하고 민감한 방법입니다. 여기에서 우리는 투과성 연구를위한 장 주머니의 준비 및 명백한 투자율 (P 응용 프로그램)의 계산을 설명 장 장벽을 가로 지르는 분자. 이 기술은 평가 약물 흡수의 방법으로 사용할 수도 있고, 위장관 질환의 동물 모델에서의 지역 상피 장벽 기능 장애를 조사.

이 프로토콜은 위장 질환의 동물 모델에서 장 장벽 기능 지역적 변화를 조사하기 위하여 사용될 수있다. 위장관 (7)의 다양한 영역에서의 점막 표면에 걸쳐 paracellular 프로브의 유량을 측정함으로써, 상피 꽉 접합 무결성을 평가할 수있다. 또한, 크기 (도 2) 또는 소수성 (도 3), 상피 섭동의 정도 또는 점막 겔층의 무결성을 paracellular 프로브?...

Watch this Scientific Journal Video about Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease at JoVE.com

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

This protocol describes the use of excised intestinal tissue preparations or "intestinal sacs" as an ex vivo model of intestinal barrier function. This model may be used to assess integrity of both the epithelial barrier and the mucous gel layer at specific intestinal sites in animal models of digestive disease.

예 생체 내 창자 주머 니나는 위장 질환 모델에서 점막 투과성을 평가하는

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying ...

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Research

JoVE Journal

Medicine

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

21.0K 조회수.  University of Newcastle. This protocol describes the use of excised intestinal tissue preparations or "intestinal sacs" as an ex vivo model of intestinal barrier function. This model may be used to assess integrity of both the epithelial barrier and the mucous gel layer at specific intestinal sites in animal models of digestive disease.

비디오: Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted in sophisticated in vitro models of the intestinal mucosa. Leveraging these emerging model systems will require adaptation of tools and techniques developed for 2D culture systems and animals. Here, we describe a technique for measuring epithelial barrier permeability in human intestinal organoids in real-time. This is accomplished by microinjection of fluorescently-labeled dextran and imaging on an inverted microscope fitted with epifluorescent filters. Real-time measurement of the barrier permeability in intestinal organoids facilitates the generation of high-resolution temporal data in human intestinal epithelial tissue, although this technique can also be applied to fixed timepoint imaging approaches. This protocol is readily adaptable for the measurement of epithelial barrier permeability following exposure to pharmacologic agents, bacterial products or toxins, or live microorganisms. With minor modifications, this protocol can also serve as a general primer on microinjection of intestinal organoids and users may choose to supplement this protocol with additional or alternative downstream applications following microinjection.

This protocol describes the measurement of epithelial barrier permeability in real-time following pharmacologic treatment in human intestinal organoids using fluorescent microscopy and live cell microscopy.

인간의 장 Organoids에 상피 장벽 침투성의 실시간 측정

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted ...

연구

발생학

Analyzing Beneficial Effects of Nutritional Supplements on Intestinal Epithelial Barrier Functions During Experimental Colitis

Inflammatory bowel diseases (IBD), including Crohn&#39;s disease and ulcerative colitis, are chronic relapsing disorders of the intestines. They cause severe problems, such as abdominal cramping, bloody diarrhea, and weight loss, in affected individuals. Unfortunately, there is no cure yet, and treatments only aim to alleviate symptoms. Current treatments include anti-inflammatory and immunosuppressive drugs that may cause severe side effects. This warrants the search for alternative treatment options, such as nutritional supplements, that do not cause side effects. Before their application in clinical studies, such compounds must be rigorously tested for effectiveness and security in animal models. A reliable experimental model is the dextran sulfate sodium (DSS) colitis model in mice, which reproduces many of the clinical signs of ulcerative colitis in humans. We recently applied this model to test the beneficial effects of a nutritional supplement containing vitamins C and E, L-arginine, and &#969;3-polyunsaturated fatty acids (PUFA). We analyzed various disease parameters and found that this supplement was able to ameliorate edema formation, tissue damage, leukocyte infiltration, oxidative stress, and the production of pro-inflammatory cytokines, leading to an overall improvement in the disease activity index. In this article, we explain in detail the correct application of nutritional supplements using the DSS colitis model in C57Bl/6 mice, as well as how disease parameters such as histology, oxidative stress, and inflammation are assessed. Analyzing the beneficial effects of different diet supplements may then eventually open new avenues for the development of alternative treatment strategies that alleviate IBD symptoms and/or that prolong the phases of remission without causing severe side effects.

Current treatments of inflammatory bowel diseases (IBD) aim at alleviating disease symptoms but may cause severe side effects. Thus, alternative strategies are being investigated in animal colitis models. Here, we explain how the beneficial effects of diet supplements on clinical IBD signs are analyzed in such a colitis model.

Inflammatory bowel diseases (IBD), including Crohn&#39;s disease and ulcerative colitis, are chronic relapsing disorders of the intestines. They cause ...

의학

An Intravital Microscopy-Based Approach to Assess Intestinal Permeability and Epithelial Cell Shedding Performance

Intravital microscopy of the gut using confocal imaging allows real time observation of epithelial cell shedding and barrier leakage in living animals. Therefore, the intestinal mucosa of anesthetized mice is topically stained with unspecific staining (acriflavine) and a fluorescent tracer (rhodamine-B dextran), mounted on a saline solution-rinsed plate and directly imaged using a confocal microscope. This technique can complement other non-invasive techniques to identify leakage of intestinal permeability, such as transmucosal passage of orally administered tracers. Besides this, the approach presented here allows the direct observation of cell shedding events at real-time. In combination with appropriate fluorescent reporter mice, this approach is suitable for shedding light into cellular and molecular mechanisms controlling intestinal epithelial cell extrusion, as well as to other biological processes. In the last decades, interesting studies using intravital microscopy have contributed to knowledge on endothelial permeability, immune cell gut homing, immune-epithelial communication and invasion of luminal components, among others. Together, the protocol presented here would not only help increase the understanding of mechanisms controlling epithelial cell extrusion, but could also be the basis for the developmental of other approaches to be used as instruments to visualize other highly dynamic cellular process, even in other tissues. Among technical limitations, optical properties of the specific tissue, as well as the selected imaging technology and microscope configuration, would in turn, determine the imaging working distance, and resolution of acquired images.

Taking advantage of intravital microscopy, the method presented here enables real-time visualization of intestinal epithelial cell shedding in living animals. Therefore, topically stained intestinal mucosa (acriflavine and rhodamineB-dextran) of anesthetized mice is imaged up to single-cell resolution using confocal microscopy.

장 내 투과성 및 상피 세포 흘리기 성능을 평가하기 위한 인트라바이탈 현미경 기반 접근법

Intravital microscopy of the gut using confocal imaging allows real time observation of epithelial cell shedding and barrier leakage in living animals. ...

면역학 및 감염병학

Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized Intestinal Loop Model

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water while preventing passage of luminal bacteria and exogenous substances. A breach of this layer results in increased permeability to luminal contents and recruitment of immune cells, both of which are hallmarks of pathologic states in the gut including inflammatory bowel disease (IBD). 
Mechanisms regulating epithelial barrier function and transepithelial migration (TEpM) of polymorphonuclear neutrophils (PMN) are incompletely understood due to the lack of experimental in vivo methods allowing quantitative analyses. Here, we describe a robust murine experimental model that employs an exteriorized intestinal segment of either ileum or proximal colon. The exteriorized intestinal loop (iLoop) is fully vascularized and offers physiological advantages over ex vivo chamber-based approaches commonly used to study permeability and PMN migration across epithelial cell monolayers.
We demonstrate two applications of this model in detail: (1) quantitative measurement of intestinal permeability through detection of fluorescence-labeled dextrans in serum after intraluminal injection, (2) quantitative assessment of migrated PMN across the intestinal epithelium into the gut lumen after intraluminal introduction of chemoattractants. We demonstrate feasibility of this model and provide results utilizing the iLoop in mice lacking the epithelial tight junction-associated protein JAM-A compared to controls. JAM-A has been shown to regulate epithelial barrier function as well as PMN TEpM during inflammatory responses. Our results using the iLoop confirm previous studies and highlight the importance of JAM-A in regulation of intestinal permeability and PMN TEpM in vivo during homeostasis and disease. 
The iLoop model provides a highly standardized method for reproducible in vivo studies of intestinal homeostasis and inflammation and will significantly enhance understanding of intestinal barrier function and mucosal inflammation in diseases such as IBD.

Dysregulated intestinal epithelial barrier function and immune responses are hallmarks of inflammatory bowel disease that remain poorly investigated due to a lack of physiological models. Here, we describe a mouse intestinal loop model that employs a well-vascularized and exteriorized bowel segment to study mucosal permeability and leukocyte recruitment in vivo.

표준화된 장 루프 모델을 사용하여 마우스에서 장 투과성 및 호중구 환전 이동의 기능 평가

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water ...

Determining Intestinal Permeability Using Lucifer Yellow in an Apical-Out Enteroid Model

Enteroids are an emerging research tool in the study of inflammatory bowel diseases such as necrotizing enterocolitis (NEC). They are traditionally grown in the basolateral-out (BO) conformation, where the apical surface of the epithelial cell faces the inner lumen. In this model, access to the luminal surface of enteroids for treatment and experimentation is challenging, which limits the ability to study host-pathogen interactions. To circumvent this, a neonatal apical-out (AO) model for necrotizing enterocolitis was created. Since intestinal epithelial cell permeability changes are pathognomonic for NEC, this protocol outlines using lucifer yellow (LY) as a marker of paracellular permeability. LY traverses the intestinal epithelial barrier via all three major paracellular pathways: pore, leak, and unrestricted. Using LY in an AO model allows for a broader study of permeability in NEC. Following IRB approval and parental consent, surgical samples of intestinal tissue were collected from human preterm neonates. Intestinal stem cells were harvested via crypt isolation and used to grow enteroids. Enteroids were grown to maturity and then transformed AO or left in BO conformation. These were either not treated (control) or were treated with lipopolysaccharide (LPS) and subjected to hypoxic conditions for the induction of in vitro NEC. LY was used to assess for permeability. Immunofluorescent staining of the apical protein zonula occludens-1 and basolateral protein &#946;-catenin confirmed AO conformation. Both AO and BO enteroids treated with LPS and hypoxia demonstrated significantly increased paracellular permeability compared to controls. Both AO and BO enteroids showed increased uptake of LY into the lumen of the treated enteroids compared to controls. The utilization of LY in an AO enteroid model allows for the investigation of all three major pathways of paracellular permeability. It additionally allows for the investigation of host-pathogen interactions and how this may affect permeability compared to the BO enteroid model.

The present protocol outlines a method that utilizes lucifer yellow in an apical-out enteroid model to determine intestinal permeability. This method can be used to determine paracellular permeability in enteroids that model inflammatory bowel diseases such as necrotizing enterocolitis.

정점 장내구 모델에서 루시퍼 옐로우를 사용한 장 투과성 측정

Enteroids are an emerging research tool in the study of inflammatory bowel diseases such as necrotizing enterocolitis (NEC). They are traditionally grown ...

Investigating Intestinal Barrier Breakdown in Living Organoids

Organoids and three-dimensional (3D) cell cultures allow the investigation of complex biological mechanisms and regulations in vitro, which previously was not possible in classical cell culture monolayers. Moreover, monolayer cell cultures are good in vitro model systems but do not represent the complex cellular differentiation processes and functions that rely on 3D structure. This has so far only been possible in animal experiments, which are laborious, time consuming, and hard to assess by optical techniques. Here we describe an assay to quantitatively determine the barrier integrity over time in living small intestinal mouse organoids. To validate our model, we applied interferon gamma (IFN-&#947;) as a positive control for barrier destruction and organoids derived from IFN-&#947; receptor 2 knock out mice as a negative control. The assay allowed us to determine the impact of IFN-&#947; on the intestinal barrier integrity and the IFN-&#947; induced degradation of the tight junction proteins claudin-2, -7, and -15. This assay could also be used to investigate the impact of chemical compounds, proteins, toxins, bacteria, or patient-derived probes on the intestinal barrier integrity.

Here we describe a technique to quantify the barrier integrity of small intestinal organoids. The fact that the method is based on living organoids enables the sequential investigation of different barrier integrity modulating substances or combinations thereof in a time-resolved manner.

살아있는 오르가노이드의 장 장벽 고장 조사

Organoids and three-dimensional (3D) cell cultures allow the investigation of complex biological mechanisms and regulations in vitro, which previously was ...

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 ...

생물학

Evaluation and Quantification of Micro Epithelial Gaps in the Colonic Mucosa using Immunofluorescence Staining

Epithelial cells lining the intestinal mucosa create a physical barrier that separates the luminal content from the interstitium. Epithelial barrier impairment has been associated with the development of various pathologies such as inflammatory bowel diseases (IBD). In the inflamed mucosa, superficial erosions or micro-erosions that corrupt epithelial monolayers correspond to sites of high permeability. Several mechanisms have been implicated in the formation of micro-erosions including cell shedding and apoptosis. These micro-erosions often represent microscopic epithelial gaps randomly distributed in the colon. Visualization and quantification of those epithelial gaps has emerged as an important tool to investigate intestinal epithelial barrier function. Here, we describe a new method to visualize the specific location of where transcellular and paracellular permeability is enhanced in the inflamed colonic mucosa. In this assay, we apply a 10 kDa fluorescent dye conjugated to a lysine fixable dextran to visualize high permeability regions (HPR) in the colonic mucosa. Additional use of cell death markers revealed that HPR encompass apoptotic foci where epithelial extrusion/shedding occurs. The protocol described here provides a simple but effective approach to visualize and quantify micro-erosions in the intestine, which is a very useful tool in disease models, in which the intestinal epithelial barrier is compromised.

<meta charset="utf8"></head><body>Evaluation and Quantification of Micro Epithelial Gaps in the Colonic Mucosa using Immunofluorescence Staining

Here, we describe a new method to visualize the specific location of where transcellular and paracellular permeability is enhanced in the inflamed colonic mucosa. In this assay, we apply a 10 kDa fluorescent dye conjugated to a lysine fixable dextran to visualize high permeability regions (HPR) in the colonic mucosa.

Epithelial cells lining the intestinal mucosa create a physical barrier that separates the luminal content from the interstitium. Epithelial barrier ...

Assessment of Gut Barrier Integrity in Mice Using Fluorescein-Isothiocyanate-Labeled Dextran

Gut barrier integrity is a hallmark of intestinal health. While gut barrier integrity can be assessed using indirect markers such as the measurement of plasma inflammatory markers and bacterial translocation to the spleen and lymph nodes, the gold standard directly quantifies the ability of selected molecules to traverse the gut mucosal layer toward systemic circulation. This article uses a non-invasive, cost-effective, and low-burden technique to quantify and follow in real time the intestinal permeability in mice using fluorescein-isothiocyanate-labeled dextran (FITC-dextran). Prior to oral supplementation with FITC-dextran, the mice are fasted. They are then gavaged with FITC-dextran diluted in phosphate-buffered saline (PBS). One hour after the gavage, the mice are subjected to general anesthesia using isoflurane, and the in vivo fluorescence is visualized in an imaging chamber. This technique aims to assess residual fluorescence in the abdominal cavity and the hepatic uptake, which is suggestive of portal migration of the fluorescent probe. Blood and stool samples are collected 4 h after oral gavage, and the mice are sacrificed. Plasma and fecal samples diluted in PBS are then plated, and the fluorescence is recorded. The concentration of FITC-dextran is then calculated using a standard curve. In previous research, in vivo imaging has shown that fluorescence rapidly spreads to the liver in mice with a weaker gut barrier induced by a low-fiber diet, while in mice supplemented with fiber to strengthen the gut barrier, the fluorescent signal is retained mostly in the gastrointestinal tract. In addition, in this study, control mice had elevated plasma fluorescence and reduced fluorescence in the stool, while inversely, inulin-supplemented mice had higher levels of fluorescence signals in the gut and low levels in the plasma. In summary, this protocol provides qualitative and quantitative measurements of intestinal permeability as a marker for gut health.

In the present study, fluorescein-isothiocyanate-labeled (FITC) dextran is administered to mice via oral gavage to evaluate intestinal permeability both in vivo and in plasma and fecal samples. As gut barrier function is affected in many disease processes, this direct and quantitative assay can be used in diverse research areas.

Fluorescein-Isothiocyanate-labeled dextran을 사용한 생쥐의 장 장벽 무결성 평가

Gut barrier integrity is a hallmark of intestinal health. While gut barrier integrity can be assessed using indirect markers such as the measurement of ...

예 생체 내 창자 주머 니나는 위장 질환 모델에서 점막 투과성을 평가하는

요약

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인간의 장 Organoids에 상피 장벽 침투성의 실시간 측정

Analyzing Beneficial Effects of Nutritional Supplements on Intestinal Epithelial Barrier Functions During Experimental Colitis

장 내 투과성 및 상피 세포 흘리기 성능을 평가하기 위한 인트라바이탈 현미경 기반 접근법

표준화된 장 루프 모델을 사용하여 마우스에서 장 투과성 및 호중구 환전 이동의 기능 평가

정점 장내구 모델에서 루시퍼 옐로우를 사용한 장 투과성 측정

살아있는 오르가노이드의 장 장벽 고장 조사

생체 외에서 그리고 Vivo에서 접근 장 상피 세포 침투성을 결정 하

Evaluation and Quantification of Micro Epithelial Gaps in the Colonic Mucosa using Immunofluorescence Staining

Evaluation and Quantification of Micro Epithelial Gaps in the Colonic Mucosa using Immunofluorescence Staining

Fluorescein-Isothiocyanate-labeled dextran을 사용한 생쥐의 장 장벽 무결성 평가