이 작품은 국립 보건원, R01CA142928의 교부금에 의해 지원되었다.

1. 마우스 측면 꼬리 정맥에 에반스 블루의 정맥 주사 <ol><li> PBS에서 에반스 파란색의 0.5 % 살균 솔루션을 준비합니다. 필요한 경우, 용해되지 않은 미립자를 제거 할 수있는 솔루션을 필터 소독. </li><li> 주사기에 200 μl 에반스 블루 솔루션을 기음. 주사기로 탈출했을 수도 모든 기포를 사용하지 마십시오. </li><li> 동물이 자유롭게 이동하지 않지만 꼬리가 처리 할 수​​ 있도록 구속 장치에 8-12주 오래된 곳 마우스. </li><li> 그래서 측면 꼬리 정맥을 쉽게 볼 수 있으며 위쪽으로 직면하고있다 그 쪽 마우스 덮개 장치를 놓습니다. </li><li> 엄지와 집게 손가락 사이의 비 지배적 인 손으로 정신 차려라. </li><li> 머리의 방향에 대한 측면 꼬리 정맥에 10 ~ 15도 각도, 베벨까지 사전에있는 바늘 (작은 게이지, 27-30)를 삽입합니다. 꼬리에 바늘과 주사기 병렬세요. </li><li> 디O이 정맥을 축소 하듯이 적절한 위치를 확인하는 압력을 다시 적용하지. </li><li> 천천히 마우스의 꼬리 정맥에 200 μl 에반스 블루 솔루션을 주입. </li><li> 플런저 진보가이 같은 정맥에 바늘의 올바른 위치의 증명되는 편의성을 관찰합니다. </li><li> 그 케이지에 다시 마우스를 넣고 30 분 동안을 관찰합니다. </li></ol> 2. 기관에서 장기 수집 및 에반스 블루의 추출 <ol><li> 자궁 경부 전위를 통해 쥐를 희생. 이 혈관 투과성과 상당한 간섭을 제한 마일 분석을 위해 자궁 경부 전위 권장합니다. 한 빨리 가능한 한 같은 시간에 모든 쥐를 희생. 죽음의 혈관이 더 융화가 될 직후에 있기 때문에, 6 생쥐 이하의 동료들과 협력합니다. </li><li> 자신의 뒤를에 마우스를 놓고 화이트 보드에 발을 고정. </li><li> 토르을 폭로 할 수있는 복부 및 흉부 캐 버티를 엽니 다acic 및 복부 기관. </li><li> 에반스 블루 넘쳐 흐름의 차이를 보여 대표 사진을 찍습니다. 모든 마우스에 동일한 조명 조건이하기 위해 같은 필드에있는 모든 쥐를 포함합니다. </li><li> 관심 장기를 수집하고 1.5 ML 튜브에 넣어 </li><li> 빈 관을 무게 0으로 균형 값을 가져. </li><li> 조직 샘플을 전송하고 무게. 모든 조직 샘플에 대한 반복합니다. 조직은 공기가 다른 장기 사이의 물 내용 변화를 제거하기 위해 건조 할 수 있습니다. </li><li> . 각 조직 샘플 튜브에 500 μl 포름 아미드를 추가합니다. </li><li> 55 ° C의 물을 욕조 또는 열 블록에 대한 모든 튜브를 전송합니다. 조직에서 에반스 블루를 추출 할 수 있도록 24-48 시간에 품다. </li></ol> 3. 에반스 블루의 정량화는 중간 조직에 Extravasated <ol><li> 펠렛 남아있는 조직 조각에 포름 아미드 / 에반스 블루 혼합물을 원심 분리기. </li><li> 610에서 흡광도를 측정NM. 빈으로 500 μl 포름 아미드를 사용합니다. </li><li> 계산 NG 에반스 블루 MG 조직 당 extravasated. </li><li> 그래프의 모든 데이터를 하겠나. </li><li> 큰 차이를 결정하기 위해 통계 분석을 수행합니다. </li></ol>

<ol>
	<li>Beck, K. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducible+NO+synthase:+role+in+cellular+signaling.">Inducible NO synthase: role in cellular signaling.</a> J. Exp. Biol. 202, 645-653 (1999).</li><li>Bertglia, S., Giusti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+nitric+oxide+in+capillary+perfusion+and+oxygen+delivery+regulation+during+systemic+hypoxia.">Role of nitric oxide in capillary perfusion and oxygen delivery regulation during systemic hypoxia.</a> Am. J. Physiol. Heart Circ. Physiol. 288, H525-H531 (2005).</li><li>Miles, A. A., Miles, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+reactions+to+histamine,+histamine-liberator+and+leutaxine+in+the+skin+of+guinea+pigs.">Vascular reactions to histamine, histamine-liberator and leutaxine in the skin of guinea pigs.</a> J. Physiol. (London). 118, 228-257 (1952).</li><li>Weis, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+permeability+in+cardiovascular+disease+and+cancer.">Vascular permeability in cardiovascular disease and cancer.</a> Curr. Opin. Hematol. 15, 243-249 (2008).</li><li>Kumar, P., Shen, Q., Pivetii, C. D., Lee, E. S., We, M. H., Yuan, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+endothelial+hyperpermeability:+implications+in+inflammation.">Molecular mechanisms of endothelial hyperpermeability: implications in inflammation.</a> Expert Rev. Mol. Med. 30, 11-19 (2009).</li><li>Viazzi, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+permeability,+blood+pressure,+and+organ+damage+in+primary+hypertension.">Vascular permeability, blood pressure, and organ damage in primary hypertension.</a> Hypertens. Res. 31, 873-879 (2008).</li><li>Scheppke, ., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+vascular+permeability+suppression+by+topical+application+of+a+novel+VEGFR2/Src+kinase+inhibitor+in+mice+and+rabbits.">Retinal vascular permeability suppression by topical application of a novel VEGFR2/Src kinase inhibitor in mice and rabbits.</a> J. Clin. Invest. 118, 2337-2346 (2008).</li><li>Blanchet, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+CD34+Leads+To+Exacerbated+Autoimmune+Arthritis+through+Increased+Vascular+Permeability.">Loss of CD34 Leads To Exacerbated Autoimmune Arthritis through Increased Vascular Permeability.</a> J. Immunol. 184, 1292-1299 (2010).</li><li>Dvorak, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mast+cell-derived+mediators+of+enhanced+microvascular+permeability,+vascular+permeability+factor/vascular+endothelial+growth+factor,+histamine,+and+serotonin,+cause+leakage+of+macromolecules+through+a+new+endothelial+cell+permeability+organelle,+the+vesiculo-vacuolar+organelle.">Mast cell-derived mediators of enhanced microvascular permeability, vascular permeability factor/vascular endothelial growth factor, histamine, and serotonin, cause leakage of macromolecules through a new endothelial cell permeability organelle, the vesiculo-vacuolar organelle.</a> Chem. Immunol. Allergy. 85, 185-204 (2005).</li><li>Le Guelte, A., Gavard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+endothelial+cell-cell+junctions+in+endothelial+permeability.">Role of endothelial cell-cell junctions in endothelial permeability.</a> Methods Mol. Biol. 763, 265-279 (2011).</li><li>Martins-Green, M., Petreaca, M., Yao, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+assay+system+for+in+vitro+detection+of+permeability+in+human+"endothelium".">An assay system for in vitro detection of permeability in human "endothelium".</a> Methods Enzymol. 443, 137-153 (2008).</li></ol>

저자는 갈등이나 경쟁 금융 이익이 없습니다.

혈관 투과성는 혈관 상태에 대한 중요한 표시입니다. 증가 혈관 투과성은 당뇨병, 고혈압, 그리고 면역 질환 6,7,8 등 여러 조직 질환에 존재하는 표시되었습니다. 증가 혈관 투과성이 전단 응력에 의해 중재 할 수 표시되었습니다 혈관 내피 성장 인자와 섬유 아세포 성장 인자, 세로토닌, 히스타민, 9 bradykinin과 같은 염증성 중재자 같은 성장 요인에 따라 결정됩니다. 물과 작은 분?...

선택적 투과성 장벽의 형성 및 유지 보수 적절한 장기 개발 및 성능 1,2에 필수적입니다. 내피 세포 라인 혈관 내강하고 혈액 모든 기관의 중간 공간 사이의 선택 전송에 필수적인 반 투과성 장벽을 형성하고 있습니다. 적절한 투과 장벽은 엄격하게 성장 요인 크린 시토 킨 및 기타 스트레스 관련 분자 세에 의해 제어됩니다 꼭 휴대 전화의 접합을 통해 유지됩니다. 내피 세포 장벽의 파괴가 증가 투자율과 혈관 누출이 발생할 수 있습니다. 이러한 효과는 다양한 질병 상태에서 볼 수 있으며 밑줄 분자 신호의 이해 학제 방법은 4,5이 필요합니다. 이 문서에서는, 우리는 마우스 모델을 사용 선박 투자율을 측정하는 생체 방법에 대해 설명합니다. 또한 마일 분석으로 알려진 우리가 설명하는 분석은 잘 establ입니다생체에 혈관 투과성을 테스트하는 방법을 ished. 검정은 기초 생리 조건 하에서, 알부민은 내피 장벽을 통과하지 않는 사실에 기반을두고 있습니다. 에반스 블루, 알부민 높은 친 화성이있는 아조 염료는 실험 동물의 혈액 스트림에 주입되고, physiologic 조건 하에서,이 혈관 내에서 제한 될 것으로 예상된다. 혈관 투과성의 자극이 추가되면, 어느 붙이기 또는 체계적으로, 혈관은 알부민에 바인딩 된 에반스 블루는 또한, 따라서 단백질을 누설하기 시작합니다. 투과성의 혈관이 조직의 급속한 푸른 착색에있는이 결과. 마우스 측면 꼬리 정맥에있는 염료를 성공적으로 주입 실험의 좋은 결과에 중요합니다. 꼬리 정맥 주입 기술은 광범위한 연습이 필요하고 실험을 시작하기 전에 마스터해야합니다. 선박 투자율은의 연령과 체중에 크게 의존imal 때문에 다른 마우스 변종을 비교하면은 마우스 또는 기타 시험 과목이 동일 출생 날짜와 무게에 가까운 있다는 필수적입니다. 내피 장벽의 투과성에 영향을 미치는 다른 요소는 온도, 습도 등의 환경 조건이며, 매우 중요한 마우스의 처리 스트레스. 실험의 결과에 영향을 미칠 수 요인의 다양한 때문에이 실험은 적어도 세 배 및 통계 분석 수행 반복되는 항상 좋습니다. 이 분석은 선박 유전자 변형 된 쥐 투자율뿐만 아니라 다른 유전 배경을 가진 쥐를 비교하거나 사용할 수 있습니다. 투자율이 변조되는 유전자의 기능에 따라 자극의 유무에 평가 될 수있다. 이 분석은 또한 혈관 투과성에 대한 서로 다른 화합물의 효과를 테스트하거나 사용할 수 있습니다.

<table border="1"><tbody><tr><td></td><td></td><td></td><td> 시약 </td></tr><tr><td> 에반스 BLUE </td><td> 시그마 </td><td> E2129 </td><td></td></tr><tr><td> 포름 아미드 </td><td> INVITROGEN </td><td> 15515-026 </td><td></td></tr><tr><td> PBS </td><td></td><td></td><td> 0.2M 인산 1.5M NaCl 산도 7.4 </td></tr><tr><td></td><td></td><td></td><td> 장비 </td></tr><tr><td> 분광 광도계 </td><td> EPPENDORF </td><td> 952000006 </td><td></td></tr><tr><td> 마우스 덮개 장치 </td><td> 하버드 장치 </td><td> 340012 </td><td></td></tr><tr><td> 주사기 </td><td> BD </td><td> 309659 </td><td></td></tr><tr><td> 바늘 </td><td> BD </td><td> 305106 </td><td> 바늘의 게이지는 동물의 크기에 따라 달라집니다. </td></tr><tr><td> 균형 </td><td> 덴버 INSTRUMENT </td><td> TP-64 </td><td></td></tr></tbody></table>

an in vivo assay to test blood vessel permeability

이 메소드는 테스트 동물 모델 쥐 에반스 블루의 정맥 주사를 기준으로합니다. 에반스 블루는 알부민 결합 염료입니다. physiologic 조건 내피는 알부민에 불 투과성이기 때문에, 에반스 블루 바운드 알부민 유적은 혈관 내에 제한. 증가 혈관 투과성 내피 세포를 촉진 pathologic 조건에서 부분적으로 자신의 가까이에 연락처를 잃게 내피는 알부민과 같은 작은 단백질 투과성이됩니다. 이 조건은 조직의 에반스 블루의 넘쳐 흐름 할 수 있습니다. 건강한 내피는 인근 vascularized의 조직에 염료의 넘쳐 흐름을 방지합니다. 증가 투자율과 기관은 그대로 내피와 장기에 비해 크게 증가 파란 착색를 표시합니다. 혈관 투과성의 수준은 간단한 시각화 또는 실험 동물 / 조직 대 제어 조직의 밀리그램 당 통합 염료의 양적 측정에 의해 평가 될 수있다. 두 포웨이 분석의 rful 측면은 단순성과 양적 특성입니다. 에반스 블루 염료는 포름 아미드의 조직의 특정 금액을 잠복기에 의해 조직에서 추출 할 수 있습니다. 에반스 블루 흡광 최대 620 나노 미터에 있으며 흡광도 최소 740 nm의에 있습니다. 에반스 블루에 대한 표준 곡선을 사용하여 광학 밀도 측정은 조직의 밀리그램 당 캡처 밀리그램 염료로 변환 할 수 있습니다. 통계 분석은 혈관 투과성에 큰 차이를 평가하기 위해 사용되어야합니다.

우리는 8~12주 기존 마우스에 혈관 누출을 테스트하기 위해 생체의 투자율 분석에를 사용했습니다. 이 테스트는 서로 다른 유전 배경의 동물 간 또는 vasculature에 영향을 미칠 치료를 받게 마우스의 단일 변종에 상대적으로 혈관 투과성을 비교에 유용합니다. 우리의 결과는 우리 실험실에서 만든 유전자 변형 쥐의 변종이 야생 형 생쥐에 비해 좀 더 투과성의 내피를 가지고 보여줍니다...

Watch this Scientific Journal Video about An in vivo Assay to Test Blood Vessel Permeability at JoVE.com

An in vivo Assay to Test Blood Vessel Permeability

우리는을 받​​게된다고?<em&gt; 생체 내</em&gt; 혈관 투과성을 테스트하는 시험. 이 분석은 간질 공간으로의 확산 염료 및 그 이후의 시각화 정맥 주사에​​ 기반을두고 있습니다.

<em&gt; 생체 내</em혈관 투과율을 테스트하려면&gt; 분석

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under ...

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An in vivo Assay to Test Blood Vessel Permeability

63.9K 조회수.  Fox Chase Cancer Center. 우리는을 받​​게된다고? 생체 내 혈관 투과성을 테스트하는 시험. 이 분석은 간질 공간으로의 확산 염료 및 그 이후의 시각화 정맥 주사에​​ 기반을두고 있습니다.

비디오: An in vivo Assay to Test Blood Vessel Permeability

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an increased risk of vascular morbidity.&#160;Yet, albeit&#160;the mounting clinical evidence, there is a paucity of clear studies of vascular toxicity and therefore the etiology of a heterogeneous group of vascular/cardiovascular disorders remains to be elucidated. Moreover, the mechanism that may underlie vascular toxicity can completely differ from the principles of chemotherapy-induced cardiotoxicity, which is related to direct myocyte injury. We have established a real-time, in vivo molecular imaging platform&#160;to evaluate&#160;the potential&#160;acute&#160;vascular toxicity of anti-cancer therapies.
We have set up a platform of in vivo, high-resolution molecular imaging&#160;in mice,&#160;suitable for visualizing vasculature within confined organs and reference blood vessels within the same individuals whereas each individual serve as its own control. Blood vessel walls were impaired after doxorubicin administration, representing a unique mechanism of vascular toxicity that may be the early event in end-organ injury. Herein, the method of fibered confocal fluorescent microscopy (FCFM) based imaging is described, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

We herein describe the method of fibered confocal fluorescent microscopy (FCFM) based imaging, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

화학 요법에 의한 혈관 독성 - 실시간<em&gt; 생체 내</em&gt; 선박 손상의 영상

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an ...

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의학

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

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying tissue compartments, restricting the transport of smaller molecules across the epithelium and almost completely prohibiting epithelial macromolecular transport. This selectivity is determined by the mucous gel layer, which limits the transport of lipophilic molecules and both the apical receptors and tight junctional protein complexes of the epithelium. In vitro cell culture models of the epithelium are convenient, but as a model, they lack the complexity of interactions between the microbiota, mucous-gel, epithelium and immune system. On the other hand, in vivo assessment of intestinal absorption or permeability may be performed, but these assays measure overall gastrointestinal absorption, with no indication of site specificity. Ex vivo permeability assays using &#34;intestinal sacs&#34; are a rapid and sensitive method of measuring either overall intestinal integrity or comparative transport of a specific molecule, with the added advantage of intestinal site specificity. Here we describe the preparation of intestinal sacs for permeability studies and the calculation of the apparent permeability (Papp) of a molecule across the intestinal barrier. This technique may be used as a method of assessing drug absorption, or to examine regional epithelial barrier dysfunction in animal models of gastrointestinal disease.

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 ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

인간 종양 이종 이식에서 마우스 세포의 고갈은 크게 대상 세포의 다운 스트림 분석을 향상

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by astrocytes, pericytes and brain endothelial cells (BECs). Here, we detail methods to assess BBB coverage using single cultures of immortalized human BECs, single cultures of primary mouse BECs, and a humanized triple culture model (BECs, astrocytes and pericytes) of the BBB. To highlight the applicability of the assays to disease states, we describe the effect of oligomeric amyloid-&#946; (oA&#946;), which is an important contributor to Alzheimer&#39;s disease (AD) progression, on BBB coverage. Further, we utilize the epidermal growth factor (EGF) to illuminate the drug screening potential of the techniques. Our results show that single and triple cultured BECs form meshwork-like structures under basal conditions, and that oA&#946; disrupts this cell meshwork formation and degenerates the preformed mesh structures, but EGF blocks this disruption. Thus, the techniques described are important for dissecting fundamental and disease-relevant processes that modulate BBB coverage.

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Maintaining blood-brain barrier coverage is key for the homeostasis of the central nervous system. This protocol describes in vitro techniques to delineate the fundamental and pathological processes that modulate blood-brain barrier coverage.

혈액 뇌 장벽 메쉬와 유사한 혈관 형성 및 분열을 평가하기위한 In vitro 시험 법

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by ...

The 4-vessel Sampling Approach to Integrative Studies of Human Placental Physiology In Vivo

The human placenta is highly inaccessible for research while still in utero. The current understanding of human placental physiology in vivo is therefore largely based on animal studies, despite the high diversity among species in placental anatomy, hemodynamics and duration of the pregnancy. The vast majority of human placenta studies are ex vivo perfusion studies or in vitro trophoblast studies. Although in vitro studies and animal models are essential, extrapolation of the results from such studies to the human placenta in vivo is uncertain. We aimed to study human placenta physiology in vivo at term, and present a detailed protocol of the method. Exploiting the intraabdominal access to the uterine vein just before the uterine incision during planned cesarean section, we collect blood samples from the incoming and outgoing vessels on the maternal and fetal sides of the placenta. When combining concentration measurements from blood samples with volume blood flow measurements, we are able to quantify placental and fetal uptake and release of any compound. Furthermore, placental tissue samples from the same mother-fetus pairs can provide measurements of transporter density and activity and other aspects of placental functions in vivo. Through this integrative use of the 4-vessel sampling method we are able to test some of the current concepts of placental nutrient transfer and metabolism in vivo, both in normal and pathological pregnancies. Furthermore, this method enables the identification of substances secreted by the placenta to the maternal circulation, which could be an important contribution to the search for biomarkers of placenta dysfunction.

The 4-vessel Sampling Approach to Integrative Studies of Human Placental Physiology In Vivo

We present a detailed method to study human placental physiology in vivo at term. The method combines blood sampling from the incoming and outgoing vessels on the maternal and fetal sides of the placenta with ultrasound measurements of volume blood flow and placental tissue sampling.

인간 태반 생리학의 통합 연구에 대한 4 혈관 샘플링 접근법<em&gt; In Vivo</em

The human placenta is highly inaccessible for research while still in utero. The current understanding of human placental physiology in vivo is therefore ...

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. To better understand the mechanism behind these surgical procedures, a rat partial liver transplantation (PLTx) model was established for relevant surgical studies. Because of the complexity of the rat PLTx model, a protocol with detailed descriptions is required. An article published previously reported a protocol in which ex vivo hepatectomy was used to achieve 50% rat PLTx. In contrast to this protocol, we introduced a re-arterialized PLTx with an in vivo 70% hepatectomy. An updated vessel-oriented hepatectomy was incorporated into the rat PLTx to refine the microsurgical procedure. The portal veins and hepatic arteries of the left lateral lobe and the median lobe were individually dissected and ligated before removal of the liver parenchyma, thereby decreasing the probability of bleeding in the remnant liver stump. Furthermore, an end-to-side vessel anastomosis between the common hepatic artery and the enlarged proper hepatic artery was introduced to re-arterialize the hepatic artery. By using this end-to-side vessel anastomosis technique, the diameter of the anastomosis was enlarged, thereby decreasing the difficulty of hand suture and maintaining a high rate of anastomotic patency. Moreover, the cuff anastomosis of the infrahepatic vena cava was slightly modified. A section of circumferential liver parenchyma around the vena cava of a recipient was preserved during cuff anastomosis to maintain the three-dimensional shape of the vascular lumen. This section of liver parenchyma was removed after completing the anastomosis. With this modification, the step involving placement of stay sutures was omitted, thereby further shortening the cuff anastomosis time. By using this protocol of rat PLTx, a low liver enzyme level, an intact liver lobular architecture and a high survival rate were achieved after microsurgery.

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Here, a protocol involving re-arterialized rat partial liver transplantation is presented. Specifically, 70% liver was resected in vivo by using an updated technique of vessel-oriented hepatectomy. The hepatic artery was reconstructed in an end-to-side manner. The cuff technique was modified to shorten the anastomosis time of the infrahepatic vena cava.

다시는 vivo에서 선박 지향 70% 쥐 부분 간 이식 Arterialized Hepatectomy

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. ...

An In Vivo Method for Evaluating the Gut-Blood Barrier and Liver Metabolism of Microbiota Products

The gut-blood barrier (GBB) controls the passage of nutrients, bacterial metabolites and drugs from intestinal lumen to the bloodstream. The GBB integrity is disturbed in gastrointestinal, cardiovascular and metabolic diseases, which may result in easier access of biologically active compounds, such as gut bacterial metabolites, to the bloodstream. Thus, the permeability of the GBB may be a marker of both intestinal and extraintestinal diseases. Furthermore, the increased penetration of bacterial metabolites may affect the functioning of the entire organism.
Commonly used methods for studying the GBB permeability are performed ex vivo. The accuracy of those methods is limited, because the functioning of the GBB depends on intestinal blood flow. On the other hand, commonly used in vivo methods may be biased by liver and kidney performance, as those methods are based on evaluation of urine or/and peripheral blood concentrations of exogenous markers. Here, we present a direct measurement of GBB permeability in rats using an in vivo method based on portal blood sampling, which preserves intestinal blood flow and is virtually not affected by the liver and kidney function. 
Polyurethane catheters are inserted into the portal vein and inferior vena cava just above the hepatic veins confluence. Blood is sampled at baseline and after administration of a selected marker into a desired part of the gastrointestinal tract. Here, we present several applications of the method including (1) evaluation of the colon permeability to TMA, a gut bacterial metabolite, (2) evaluation of liver clearance of TMA, and (3) evaluation of a gut-portal blood-liver-peripheral blood pathway of gut bacteria-derived short-chain fatty acids. Furthermore, the protocol may also be used for tracking intestinal absorption and liver metabolism of drugs or for measurements of portal blood pressure.

An In Vivo Method for Evaluating the Gut-Blood Barrier and Liver Metabolism of Microbiota Products

The access of nutrients, microbiota metabolites and medicines to the circulation is controlled by the gut-blood barrier (GBB). We describe a direct method for measuring the GBB permeability in vivo, which, in contrast to commonly used indirect methods, is virtually not affected by liver and kidney functions.

창 자-혈액 장벽 및 간 대사 Microbiota 제품의 평가 위한 Vivo에서 방법

The gut-blood barrier (GBB) controls the passage of nutrients, bacterial metabolites and drugs from intestinal lumen to the bloodstream. The GBB integrity ...

Using an Automated Hirschberg Test App to Evaluate Ocular Alignment

A smartphone app has been developed to perform the automated photographic Hirschberg test for objective measurement of ocular misalignment. By computing the difference in corneal reflection generated by the phone camera flash relative to the iris center based on high resolution images, the app can measure misalignment with a much higher precision than the naked eye performing the Hirschberg test. It has been validated in a previous clinical evaluation study by comparing to the clinical gold standard-prism and alternate cover test. The goal of this article is to describe the testing techniques regarding how to use the app to measure ocular alignment for different fixation distances, without or with cover to break fusion, as well as angle kappa, so that users can use the app to perform equivalent tests typically done in clinic using prisms.

We present a protocol of using a smartphone app to perform Hirschberg test for measuring manifest and intermittent ocular misalignment (strabismus) under near and far fixation conditions.

안구 정렬을 평가하기 위해 자동화 된 Hirschberg 테스트 응용 프로그램을 사용하여

A smartphone app has been developed to perform the automated photographic Hirschberg test for objective measurement of ocular misalignment. By computing ...

Measurement of Strial Blood Flow in Mouse Cochlea Utilizing an Open Vessel-Window and Intravital Fluorescence Microscopy

Transduction of sound is metabolically demanding, and the normal function of the microvasculature in the lateral wall is critical for maintaining endocochlear potential, ion transport, and fluid balance. Different forms of hearing disorders are reported to involve abnormal microcirculation in the cochlea. Investigation of how cochlear blood flow (CoBF) pathology affects hearing function is challenging due to the lack of feasible interrogation methods and the difficulty in accessing the inner ear. An open vessel-window in the lateral cochlear wall, combined with fluorescence intravital microscopy, has been used for studying CoBF changes in vivo, but mostly in guinea pig and only recently in the mouse. This paper and the associated video describe the open vessel-window method for visualizing blood flow in the mouse cochlea. Details include 1) preparation of the fluorescent-labeled blood cell suspension from mice; 2) construction of an open vessel-window for intravital microscopy in an anesthetized mouse, and 3) measurement of blood flow velocity and volume using an offline recording of the imaging. The method is presented in video format to show how to use the open window approach in mouse to investigate structural and functional changes in the cochlear microcirculation under normal and pathological conditions.

An open vessel-window approach using fluorescent tracers provides sufficient resolution for cochlear blood flow (CoBF) measurement. The method facilitates the study of structural and functional changes in CoBF in mouse under normal and pathological conditions.

열린 혈관 창과 생체 내 형광 현미경을 사용한 마우스 달팽이관의 심방 혈류 측정

Transduction of sound is metabolically demanding, and the normal function of the microvasculature in the lateral wall is critical for maintaining ...

Precision Cut Lung Slices as an Efficient Tool for Ex vivo Pulmonary Vessel Structure and Contractility Studies

The visualization of murine lung tissue provides valuable structural and cellular information regarding the underlying airway and vasculature. However, the preservation of pulmonary vessels that truly represents physiological conditions still presents challenges. In addition, the delicate configuration of murine lungs result in technical challenges preparing samples for high-quality images that preserve both cellular composition and architecture. Similarly, cellular contractility assays can be performed to study the potential of cells to respond to vasoconstrictors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these technical issues, the precision-cut lung slice (PCLS) method can be applied as an efficient alternative to visualize lung tissue in three dimensions without regional bias and serve as a live surrogate contractility model for up to 10 days. Tissue prepared using PCLS has preserved structure and spatial orientation, making it ideal to study disease processes ex vivo. The location of endogenous tdTomato-labeled cells in PCLS harvested from an inducible tdTomato reporter murine model can be successfully visualized by confocal microscopy. After exposure to vasoconstrictors, PCLS demonstrates the preservation of both vessel contractility and lung structure, which can be captured by a time-lapse module. In combination with the other procedures, such as western blot and RNA analysis, PCLS can contribute to the comprehensive understanding of signaling cascades that underlie a wide variety of disorders and lead to a better understanding of the pathophysiology in pulmonary vascular diseases.

Precision Cut Lung Slices as an Efficient Tool for Ex vivo Pulmonary Vessel Structure and Contractility Studies

Presented here is a protocol for preserving the vascular contractility of PCLS murine lung tissue, resulting in a sophisticated three-dimensional image of the pulmonary vasculature and airway, which can be preserved for up to 10 days that is susceptible to numerous procedures.

정밀 절단 폐 슬라이스는 전 생체 폐 혈관 구조 및 수축 연구를위한 효율적인 도구로

The visualization of murine lung tissue provides valuable structural and cellular information regarding the underlying airway and vasculature. However, ...