이 작품은 미국 심장 협회 (EIA 0740129N)와 NIH T32 HL90610에 의해 지원되었다.

<ol>
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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+of+extraparenchymal+resistance+arteries+of+rat+skeletal+muscle+to+reduce+PO2.">Response of extraparenchymal resistance arteries of rat skeletal muscle to reduce PO2.</a> Am. J. Physiol. 267, H706-H715 (1994).</li><li>Durand, M. J., Raffai, G., Weinberg, B. D., Lombard, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiotensin-(1-7)+and+low-dose+angiotensin+II+infusion+reverse+salt-induced+endothelial+dysfunction+via+different+mechanisms+in+rat+middle+cerebral+arteries.">Angiotensin-(1-7) and low-dose angiotensin II infusion reverse salt-induced endothelial dysfunction via different mechanisms in rat middle cerebral arteries.</a> Am. J. Physiol. Heart Circ. Physiol. 299, H1024-H1033 (2010).</li><li>LeBlanc, A. J., Cumpston, J. L., Chen, B. T., Frazer, D., Castranova, V., Nurkiewicz, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoparticle+inhalation+impairs+endothelium-dependent+vasodilation+in+subepicardial+arterioles.">Nanoparticle inhalation impairs endothelium-dependent vasodilation in subepicardial arterioles.</a> J. Toxicol. Environ. Health A. 72, 1576-1584 (2009).</li><li>Jernigan, N. L., LaMarca, B., Speed, J., Galmiche, L., Granger, J. P., Drummond, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dietary+salt+enhances+benzamil-sensitive+component+of+myogenic+constriction+in+mesenteric+arteries.">Dietary salt enhances benzamil-sensitive component of myogenic constriction in mesenteric arteries.</a> Am. J. Physiol. Heart Circ. Physiol. 294, H409-H420 (2008).</li><li>Stapleton, P. A., Goodwill, A. G., James, M. E., Frisbee, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+mechanisms+of+endothelium-dependent+dilation+in+skeletal+muscle+arterioles+with+genetic+hypercholesterolemia.">Altered mechanisms of endothelium-dependent dilation in skeletal muscle arterioles with genetic hypercholesterolemia.</a> Am. J. Physiol. Regul. Integr. Comp. Physiol. 293, R1110-R1119 (2007).</li><li>Goodwill, A. G., Stapleton, P. A., James, M. E., d'Audiffret, A. C., Frisbee, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+arachidonic+acid-induced+thromboxane+generation+impairs+skeletal+muscle+arteriolar+dilation+with+genetic+dyslipidemia.">Increased arachidonic acid-induced thromboxane generation impairs skeletal muscle arteriolar dilation with genetic dyslipidemia.</a> Microcirculation. 15, 621-631 (2008).</li><li>Baumbach, G. L., Hadju, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanics+and+composition+of+cerebral+arterioles+in+renal+and+spontaneously+hypertensive+rats.">Mechanics and composition of cerebral arterioles in renal and spontaneously hypertensive rats.</a> Hypertension. 21, 816-826 (1993).</li><li>Uchida, E., Bohr, D. F., Hoobler, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+studying+isolated+resistance+vessel+from+rabbit+mesentery+and+brain+and+their+responses+to+drugs.">A method for studying isolated resistance vessel from rabbit mesentery and brain and their responses to drugs.</a> Circ. Res. 4, 525-536 (1967).</li><li>Davis, M. J., Kuo, L., Chilian, W. M., Muller, J. M. I. s. o. l. a. t. e. d., Barker, J. H., Anderson, G. L., Menger, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+23.">Chapter 23.</a> Isolated, perfused microvessels. In: Clinically Applied Microcirculation Research. 32, 435-456 (1995).</li><li>Lombard, J. H., Liu, Y., Fredricks, K. T., Bizub, D. M., Roman, R. J., Rusch, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+and+mechanical+responses+of+rat+middle+cerebral+arterieal+to+reduced+PO2+and+prostacyclin.">Electrical and mechanical responses of rat middle cerebral arterieal to reduced PO2 and prostacyclin.</a> Am. J. Physiol. 276, H509-H516 (1994).</li></ol>

관심의 어떠한 충돌 선언 없습니다.

이러한 일반적인 기술은 쉽게 대부분의 조직에 적용할 수 있지만 제시 프로토콜, 골격 근육 microvessel의 분리, 제거 및 이중 cannulation을 설명합니다. 현재 원고 내용은 용어 &quot;arteriole&quot;도 주요 장기에 재관류 저항의 규정에 기여 아니면 휴식 활성 톤 미만의 직경 70-120 μm의 사이에 이르기까지 저항 혈관을 설명하기 위해 저자가 사용되었습니다 조직. 일부 수정이 시스템은...

<table border="1"><tbody><tr><td> 시약 및 장비 </td><td> 회사 </td><td> 덧글 / 카탈로그 # </td></tr><tr><td> 선박 상공 회의소 </td><td> 사용자 정의 </td><td> 데이브 Eick (MCW) </td></tr><tr><td> 온수 순환 물 목욕 </td><td> PolyScience 및 Haake </td><td> Haake DC 10 </td></tr><tr><td> Pipets </td><td> 프레데릭 Haer &amp; (주) </td><td> 모세관 튜브 2.0 mm OD X 1.0 mm ID (27-33-1) </td></tr><tr><td> 압력 모니터 </td><td> 세계 정밀 계측기 </td><td></td></tr><tr><td> 물의 외피 저수지 </td><td> 사용자 정의 </td><td></td></tr><tr><td> 외부 광원 </td><td> 세계 정밀 계측기 </td><td> Novaflex </td></tr><tr><td> Pipet의 풀러 </td><td> MicroData 인 스트 루먼트 </td><td> PMP102 Micropipet 풀러 </td></tr><tr><td> 전체 결점수술 도구, 표준 </td><td> 파인 과학 도구 </td><td> 뒤몽 </td></tr><tr><td> 울트라 파인 집게 </td><td> 파인 과학 도구 </td><td> Inox # 5 </td></tr><tr><td> 실크 봉합사 스레드 </td><td> Ethilon </td><td> # 10-0 또는 9-0 </td></tr><tr><td> 스테레오 현미경 </td><td> 올림포스 산 </td><td> 올림푸스 SZ-11 </td></tr><tr><td> 아날로그 비디오 캘리퍼스 </td><td> Boeckeler </td><td> 비아 컨트롤러 (비아-100) </td></tr><tr><td> 고해상도 아날로그 카메라 </td><td> 파나소닉 </td><td> GP-MF 602 </td></tr><tr><td> 산소 탱크 </td><td> 지역 </td><td> 21 % 밸런스 질소와 5 %는이 밸런스 질소를 공동 </td></tr><tr><td> 관류 </td><td> Tygon </td><td></td></tr><tr><td> 펌프 배수 </td><td> 콜 Parmer 악기 (주) </td><td></td></tr><tr><td> 수정된 생쥐 PSS </td><td> 아래의 제조법을 참조하십시오 </td><td></TD&gt; </tr><tr><td> 반 Breemen의 이완 PSS </td><td> 아래의 제조법을 참조하십시오 </td><td></td></tr></tbody></table> 표 1. 수치로 표시 절연 microvessel 스테이션 설정의 주요 구성 요소의 목록입니다. <table border="1"><tbody><tr><td> 수정된 생쥐 PSS 제조법 </td><td> PSS 2 리터를 만들려면 </td><td> 20X 소금 증권 (2L) </td><td> 20X 버퍼 증권 (2L) </td></tr><tr><td> NaCl </td><td></td><td> 278.0 g </td><td></td></tr><tr><td> KCl </td><td></td><td> 14.0 g </td><td></td></tr><tr><td> MgSO 4 7H 2 O </td><td></td><td> 11.5 g </td><td></td></tr><tr><td> CaCl 2-H 2 O </td><td></td><td> 9.4 g </td><td></td></tr><tr> &lt;TD&gt; NaHCO 3 </td><td></td><td></td><td> 80.8 g </td></tr><tr><td> EDTA (에틸렌 다이아 민 테트라 초산) </td><td></td><td></td><td> 0.4 g </td></tr><tr><td> 아뇨 2 PO 4 </td><td> 0.28 g </td><td></td><td></td></tr><tr><td> 포도당 </td><td> 1.98 g </td><td></td><td></td></tr><tr><td> 20x 소금 증권 </td><td> 100 ML </td><td></td><td></td></tr><tr><td> 20x 버퍼 증권 </td><td> 100 ML </td><td></td><td></td></tr><tr><td> 증류수 </td><td> 1800 ML </td><td></td><td></td></tr></tbody></table> 표 2. 격리된 microvessel 프로토콜에서 사용되는 표준 생리 식염수 (PSS)를위한 레시피. 레시피에 대한 의견 : 소금 주식 2 패와 버퍼 증권 2 패하십시오. 이 C사용하지 않는 경우 냉장 있겠지만 잘 종종 PSS를 준비하기 전에 그들을 흔들어. 추가 재료는 최종 PSS의 준비 시점에 추가됩니다. <table border="1"><tbody><tr><td> 반 Breemen의 이완 PSS </td><td> PSS 2 리터를 만들려면 </td><td> 20X 소금 증권 (1L) </td><td> 20X 버퍼 증권 (1L) </td></tr><tr><td> NaCl </td><td></td><td> 107.4 g </td><td></td></tr><tr><td> KCl </td><td></td><td> 7.0 g </td><td></td></tr><tr><td> MgSO 4 7H 2 O </td><td></td><td> 5.76 g </td><td></td></tr><tr><td> MgCl 2 6H 2 O </td><td></td><td> 81.32 g </td><td></td></tr><tr><td> NaHCO 3 </td><td></td><td></td><td> 40.4 g </td></tr><tr><td> 동부 서머 타임</td><td></td><td></td><td> 0.2 g </td></tr><tr><td> EGTA </td><td></td><td></td><td> 15.22 </td></tr><tr><td> 아뇨 2 PO 4 </td><td> 0.28 g </td><td></td><td></td></tr><tr><td> 포도당 </td><td> 1.98 g </td><td></td><td></td></tr><tr><td> 20x 소금 증권 </td><td> 100 ML </td><td></td><td></td></tr><tr><td> 20x 버퍼 증권 </td><td> 100 ML </td><td></td><td></td></tr><tr><td> 증류수 </td><td> 1800 ML </td><td></td><td></td></tr></tbody></table> 표 3. 제로 활성 톤의 조건 하에서 절연 microvessel 프로토콜에 사용되는 반 Breemen의 이완 생리 식염수 (PSS)를위한 레시피. 레시피에 대한 의견 : 소금 증권 1 패 만들기버퍼 증권의 ND 1 패. 이들은 사용하지 않는 경우 냉장 있겠지만 잘 종종 PSS를 준비하기 전에 그들을 흔들 수있다. 추가 재료는 최종 이완 PSS의 준비 시점에 추가됩니다.

the ex vivo isolated skeletal microvessel preparation for investigation of vascular reactivity

절연 microvessel 준비는, 따라서 관류 저항 1-5 여러 요인 공헌 제어하는 혈관 직경의 전형을 허용 전직 생체내 준비이며. 이것은 처음. 15 몇 년 전 우치다 외 의해 설명, 대형 측정에서였다 고전 실험 준비입니다. 이것은 초기 설명은 광범위 주로 버지니아 6-8의 대학에서 박사 브라이언 Duling의 실험실에서 수정되고 향상되었습니다 기법에 대한 기초를 제공하고, 우리는 다음 페이지에서 현재의 접근 방식을 제시. 이러한 준비는 특히 선택의 microvessel 같은 시궁창에서 gracilis의 arteriole을 참조되지만, 기본적인 준비가 쉽게 종의 9-13 전역의 거의 모든 다른 조직이나 기관으로부터 격리 선박에 적용할 수 있습니다. 고립된 microvessels의 기계식 (즉, 차원) 변경 사항을 쉽게 평가할 수생리학 (예 : hypoxia, intravascular 압력, 또는 전단) 또는 pharmacological 도전, 그리고 손상에 통합 반응 구성된 기계론의 요소로 통찰력을 제공할 수 있지만 예전의 생체내, 조직의 광범위한 배열에 대응 인치 이 방법의 중요성 또한 intravascular 압력 (myogenic), 자율 innervation, hemodynamic 포함한 기타 출처로부터 기부금의 많은 컨트롤을 허용하는 동시에 그것이, microvessel 직경의 통합 규제에 영향 손쉬운 조작을 허용하는 것입니다 ( 예를 들어, 전단 응력), 내피 의존하거나 독립적인 자극, 호르몬, 그리고 parenchymal 영향, 부분적인 목록을 제공합니다. 적절한 실험 조건에서하고 적절한 목표, 이것은 생체내 또는 쉽게 광범위한 체계적인 변수의 손쉬운 제어를 허용하지 않는 현장 조직 / 기관의 준비에서 이상의 장점이 될 수 있습니다. mA이 준비의 조 제한은 본질적으로 그 강점의 결과입니다. 정의함으로써, 이들 선박의 동작이 혈관 저항의 규정에 가장 중요한 참여자의 많은과 같은 신경 체액, 신진 대사 등을 포함하여 제거된 조건에서 연구되고 수사관들은 피하기 위해 오버 경고합니다 통역이 준비를 활용 수집되는 데이터의 추정. 이러한 준비에 관해서는 관심의 또 다른 중요한 영역이 같은 내피 안감이나 혈관 평활근 세포와 같은 구성 요소를 손상하는 것은 매우 쉬울 수있다는 것입니다, 오류 등 그 변수 소스를 소개하실 수 있습니다. 그것은 강력하게 개인 조사는 실험 시작시와 정기적으로 프로토콜의 과정에 걸쳐 모두, 준비의 품질을 보장​​하기위한 적절한 측정을 활용하는 것이 좋습니다.

Watch this Scientific Journal Video about The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity at JoVE.com

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

<em&gt; 예 생체내</em&gt; 준비는 혈관 vasoactive 자극에 반응하며 수동 벽 기계를 통해 기본적인 구조적 특성의 평가 모두의 심문에 대한 가장 큰 gracilis 근육 저항 arterioles의 분리에 대해 설명되어 있습니다.

예 생체내</em혈관 반응성 조사를위한> 절연 골격 Microvessel 준비

The isolated  microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of  factors that control vessel ...

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Biology

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

10.6K 조회수.  West Virginia University.  예 생체내 준비는 혈관 vasoactive 자극에 반응하며 수동 벽 기계를 통해 기본적인 구조적 특성의 평가 모두의 심문에 대한 가장 큰 gracilis 근육 저항 arterioles의 분리에 대해 설명되어 있습니다.

비디오: The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of imaging had invented. While many macroscopic parameters of blood flow reflect flow velocity, changes in blood flow velocity and red blood cell (RBC) flux does not hold linear relationship in the microscopic observations. There are reports of discrepancy between RBC velocity and RBC flux, RBC flux and plasma flow volume, and of spatial and temporal heterogeneity of flow regulation in the peripheral tissues in microscopic observations, a scientific basis for the requirement of more detailed studies in microcirculatory regulation using intravital microscopy. METHODS: We modified Jeff Lichtman's method of in vivo microscopic observation of mouse sternomastoid muscles. Mice are anesthetized,
ventilated, and injected with PKH26L-fluorescently labeled RBCs for microscopic observation.RESULT &amp; CONCLUSIONS: Fluorescently labeled RBCs are detected and distinguished well by a wide-field microscope. Muscle contraction evoked by electrical stimulation induced increase in RBC flux. Quantification of other parameters including RBC velocity and capillary density were feasible. Mice tolerated well the surgery, injection of stained RBCs, microscopic observation, and electrical stimulation. No muscle or blood vessel damage was observed, suggesting that our method is relatively less invasive and suited for long-term observations.

A new versatile method for observation of microcirculation is presented. It is considered suitable for long-term observation, and for combination with pharmacophysiological or molecular biological interventions.

내부 핵심 현미경에 의해 골격근의 생체내 미세 순환 측정

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of ...

연구

생물학

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

Lentiviral - 중재 최저 동안 예 VIVO</em인간 조혈 줄기 세포의> 적혈구 조혈

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, fatigability, and recovery after fatigue can be obtained to assess specific aspects of the excitation-contraction coupling (ECC) process such as excitability, contractile machinery and Ca2+ handling ability. This method removes the nerve and blood supply and focuses on the isolated skeletal muscle itself. We routinely use this method to identify genetic components that alter the contractile property of skeletal muscle though modulating Ca2+ signaling pathways. Here, we describe a newly identified skeletal muscle phenotype, i.e., mechanic alternans, as an example of the various and rich information that can be obtained using the in vitro muscle contractility assay. Combination of this assay with single cell assays, genetic approaches and biochemistry assays can provide important insights into the mechanisms of ECC in skeletal muscle.

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

We describe a method to directly measure muscle force, muscle power, contractile kinetics and fatigability of isolated skeletal muscles in an in vitro system using field stimulation. Valuable information on Ca2+ handling properties and contractile machinery of the muscle can be obtained using different stimulating protocols.

절연 골격근의 수축성, Fatigability 및 Alternans의 예 VIVO 평가

 
Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

소설<em&gt; 생체</em배아 마우스 사랑을&gt; 문화 방법

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.

The isolated blood-perfused lung preparation makes it feasible to visualize microvessel networks on the lung surface. Here we describe an approach to quantify permeability of single microvessels in isolated lungs using real time fluorescence imaging.

고립 된 혈액 관류 쥐 폐 준비에 단일 미세 혈관 투과성의 양을

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...

Contractility Measurements on Isolated Papillary Muscles for the Investigation of Cardiac Inotropy in Mice

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The contractile characteristics can be evaluated independently of external influences such as vascular tonus or neurohumoral status. It depicts a scientific approach between single cell measurements with isolated cardiac myocytes and in vivo studies like echocardiography. Thus, papillary muscle preparations serve as an excellent model to study cardiac physiology/pathophysiology and can be used for investigations like the modulation by pharmacological agents or the exploration of transgenic animal models. Here, we describe a method of isolating the murine left anterior papillary muscle to investigate cardiac contractility in an organ bath setup. In contrast to a muscle strip preparation isolated from the ventricular wall, the papillary muscle can be prepared in toto without damaging the muscle tissue severely. The organ bath setup consists of several temperature-controlled, gassed and electrode-equipped organ bath chambers. The isolated papillary muscle is fixed in the organ bath chamber and electrically stimulated. The evoked twitch force is recorded using a pressure transducer and parameters such as twitch force amplitude and twitch kinetics are analyzed. Different experimental protocols can be performed to investigate the calcium- and frequency-dependent contractility as well as dose-response curves of contractile agents such as catecholamines or other pharmaceuticals. Additionally, pathologic conditions like acute ischemia can be simulated.

Murine left ventricular papillary muscle can be used to investigate cardiac contractility in vitro. This article describes in detail the isolation and experimental protocols to study cardiac contractile characteristics.

마우스의 심장 수축력의 조사에 대 한 격리 유두 근육의 수축력 측정

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The ...

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, is expressed in neurons in response to various stimuli. The protein product can be readily detected with immunohistochemical techniques leading to the use of c-FOS detection to map groups of neurons that display changes in their activity. In this article, we focused on the identification of brainstem neuronal populations involved in the ventilatory adaptation to hypoxia or hypercapnia. Two approaches were described to identify involved neuronal populations in vivo in animals and ex vivo in deafferented brainstem preparations. In vivo, animals were exposed to hypercapnic or hypoxic gas mixtures. Ex vivo, deafferented preparations were superfused with hypoxic or hypercapnic artificial cerebrospinal fluid. In both cases, either control in vivo animals or ex vivo preparations were maintained under normoxic and normocapnic conditions. The comparison of these two approaches allows the determination of the origin of the neuronal activation i.e., peripheral and/or central. In vivo and ex vivo, brainstems were collected, fixed, and sliced into sections. Once sections were prepared, immunohistochemical detection of the c-FOS protein was made in order to identify the brainstem groups of cells activated by hypoxic or hypercapnic stimulations. Labeled cells were counted in brainstem respiratory structures. In comparison to the control condition, hypoxia or hypercapnia increased the number of c-FOS labeled cells in several specific brainstem sites that are thus constitutive of the neuronal pathways involved in the adaptation of the central respiratory drive.

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Here, we present a protocol based on c-FOS protein immunohistological detection, a classical technique used for the identification of neuronal populations involved in specific physiological responses in vivo and ex vivo.

은 C-FOS 단백질 면역 조직 검색 : 특정 생리적 반응에 관여 중앙 진학의 마커로서 유용한 도구<em&gt; 생체</em&gt; 및<em&gt; 전의 VIVO</em

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, ...

Retinal Cryo-sections, Whole-Mounts, and Hypotonic Isolated Vasculature Preparations for Immunohistochemical Visualization of Microvascular Pericytes

Retinal pericytes play an important role in many diseases of the eye. Immunohistochemical staining techniques of retinal vessels and microvascular pericytes are central to ophthalmological research. It is vital to choose an appropriate method of visualizing the microvascular pericytes. We describe retinal microvascular pericyte immunohistochemical staining in cryo-sections, whole-mounts, and hypotonic isolated vasculature using antibodies for platelet-derived growth factor receptor &#946; (PDGFR&#946;) and nerve/glial antigen 2 (NG2). This allows us to highlight advantages and shortcomings of each of the three tissue preparations for the visualization of the retinal microvascular pericytes. Cryo-sections provide transsectional visualization of all retinal layers but contain only a few occasional transverse cuts of the microvasculature. Whole-mount provides an overview of the entire retinal vasculature, but visualization of the microvasculature can be troublesome. Hypotonic isolation provides a method to visualize the entire retinal vasculature by the removal of neuronal cells, but this makes the tissue very fragile.

We demonstrate three different tissue preparation techniques for immunohistochemical visualization of rat retinal microvascular pericytes, i.e., cryo-sections, whole-mounts, and hypotonic isolation of the vascular network.

망막 Cryo-섹션, Microvascular Pericytes의 Immunohistochemical 시각화에 대 한 전체-마운트 및 소형 고립 된 맥 관 구조

Retinal pericytes play an important role in many diseases of the eye. Immunohistochemical staining techniques of retinal vessels and microvascular ...

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

The retina is a highly metabolically active tissue that requires a substantial blood supply. The retinal circulation supports the inner retina, while the choroidal vessels supply the photoreceptors. Alterations in retinal perfusion contribute to numerous sight-threatening disorders, including diabetic retinopathy, glaucoma and retinal branch vein occlusions. Understanding the molecular mechanisms involved in the control of blood flow through the retina and how these are altered during ocular disease could lead to the identification of new targets for the treatment of these conditions. Retinal arterioles are the main resistance vessels of the retina, and consequently, play a key role in regulating retinal hemodynamics through changes in luminal diameter. In recent years, we have developed methods for isolating arterioles from the rat retina which are suitable for a wide range of applications including cell physiology studies. This preparation has already begun to yield new insights into how blood flow is controlled in the retina and has allowed us to identify some of the key changes that occur during ocular disease. In this article, we describe methods for the isolation of rat retinal arterioles and include protocols for their use in patch-clamp electrophysiology, calcium imaging and pressure myography studies. These vessels are also amenable for use in PCR-, western blotting- and immunohistochemistry-based studies.

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

This manuscript describes a straightforward protocol for the isolation of arterioles from the rat retina that can be used in electrophysiological, calcium imaging and pressure myography studies.

Ex Vivo 세포 생리학 연구에 대 한 망막 Arterioles의 절연

The retina is a highly metabolically active tissue that requires a substantial blood supply. The retinal circulation supports the inner retina, while the ...

Assessment of Vascular Tone Responsiveness using Isolated Mesenteric Arteries with a Focus on Modulation by Perivascular Adipose Tissues

Altered vascular tone responsiveness to pathophysiological stimuli contributes to the development of a wide range of cardiovascular and metabolic diseases. Endothelial dysfunction represents a major culprit for the reduced vasodilatation and enhanced vasoconstriction of arteries. Adipose (fat) tissues surrounding the arteries play important roles in the regulation of endothelium-dependent relaxation and/or contraction of the vascular smooth muscle cells. The cross-talks between the endothelium and perivascular adipose tissues can be assessed ex vivo using mounted blood vessels by a wire myography system. However, optimal settings should be established for arteries derived from animals of different species, ages, genetic backgrounds and/or pathophysiological conditions.

The protocol describes the use of wire myography to evaluate the transmural isometric tension of mesenteric arteries isolated from mice, with special consideration of the modulation by factors released from endothelial cells and perivascular adipose tissues.

예 생체내 절연 골격 Microvessel 준비

요약

더 많은 비디오 탐색

이 비디오의 챕터

내부 핵심 현미경에 의해 골격근의 생체내 미세 순환 측정

Lentiviral - 중재 최저 동안 예 VIVO 적혈구 조혈

절연 골격근의 수축성, Fatigability 및 Alternans의 예 VIVO 평가

소설

고립 된 혈액 관류 쥐 폐 준비에 단일 미세 혈관 투과성의 양을

마우스의 심장 수축력의 조사에 대 한 격리 유두 근육의 수축력 측정

은 C-FOS 단백질 면역 조직 검색 : 특정 생리적 반응에 관여 중앙 진학의 마커로서 유용한 도구

망막 Cryo-섹션, Microvascular Pericytes의 Immunohistochemical 시각화에 대 한 전체-마운트 및 소형 고립 된 맥 관 구조

Ex Vivo 세포 생리학 연구에 대 한 망막 Arterioles의 절연

혈관 주위 지방 조직에 의해 변조에 초점을 맞춘 분리 된 장 간 동맥을 사용 하 여 혈관 톤 응답성의 평가