우리는 재정 지원을 위해 스콧과 백인 병원 연구 보조금 프로그램을 인정하고 텍사스 A &amp; 공 초점 레이저 현미경의 사용에 대한 의학 통합 이미징 연구소의 M 건강 과학 센터 대학. 우리는 원고 편집에 대한 도움말 씨 글렌 Cryer을 인정합니다.

내피 세포의 1. 시드 <ol><li> 성인 스프 라그 돌리 쥐에서 RBMEC의의 차 문화를 얻습니다 (또는 상업적를 얻을). </li><li> 100cm의 피브로넥틴 (50 μg의 / ㎖) 래트 뇌 내피 세포 성장 배지를 사용하여 코팅 된 배양 접시에서 배양을 RBMECs. 포화 상태에 도달 할 때까지, 매 2 일 매체를 변경합니다. </li><li> 80-90%의 포화 상태에 도달에서 부드럽게 소용돌이 5 ml의 인산염 완충 식염수 (PBS)에있는 세포를 씻으십시오. 세포를 37 ° C로 평형화 따뜻한 0.25 % 트립신 에틸렌 디아민 테트라 아세트산 (EDTA) 용액 1 ㎖, 그들을 노출시킴으로써 분리된다. </li><li> 세포를 분리 및 분산 될 때까지 2-5 분 동안 37 ℃에서 세포를 배양한다. 참고 : 세포를 분리 배양 접시를 누릅니다. 현미경보기 세포 접시의 표면으로부터 세포의 완전한 분리를 확인하기 위해. </li><li> 트립신을 중화하기 위해 페트리 접시에 5 ml의 완전한 미디어를 추가합니다.분리 된 세포를 함유하는 배지를 피펫 및 15 mL의 원심 분리 튜브로 수집. . </li><li> 5 분 동안 220 XG에서 내피 세포를 포함하는 미디어를 원심 분리기. </li><li> 뜨는을 대기음 후 자동 세포 계수기 또는 혈구를 사용하여 계산됩니다 피펫 세포와 부드럽게를 혼합하고 아래로 3-5 ml의 신선한 쥐의 뇌 내피 세포 성장 매체에 cells.Suspend에게 펠렛을 함유 펠렛을 유지합니다. </li><li> 물론 당 10,000-15,000 세포 사이의 범위 파종 밀도 / 웰, 8 아니라 멸균 챔버 슬라이드 시스템 프리 코트 피브로넥틴 (50 μg의 / ㎖)에 0.7 cm (2) 세포 현탁액을 전송합니다. 합류가 될 때까지 37 ℃에서 세포를 성장 주 : 실험에 의해 필요에 따라 웨스턴 블롯 또는 다른 실험을 수행하기 위해, 셀은 10cm의 세포 배양 접시 나 특별한 요리에서 성장 될 수있다. </li></ol> 2. 산소와 체외에서 포도당 박탈 - 재산 소화모델 참고 : 다음 프로토콜 Zulueta 등의 알에서 적응하고있다. 1997; 주홍 H 등. 2012 8,9. <ol><li> 에 RBMECs에 OGD-R의 효과를 (그림 1 참조) 공부 저산소 세포 배양 시스템을 사용합니다. 설치 및 제조자의 지시에 따라서, 실험을 시작하기 전에 저산소증 세포 배양 시스템을 교정. </li><li> 37 ° C 배양기에서 합류 챔버 슬라이드 (단계 1.8)를 제거합니다. 탈산 소화와 챔버 슬라이드에서 완전 배지를 교체에는 글루코스, 둘 베코 변형 이글 배지 (DMEM), 37 ° C에서 2 시간 동안 95 %, N (2) 및 5 % CO 2로 저산소증 챔버 내에 배치는 OGD 상태를 나타내는 없음. </li><li> 37 ° C에서 95 % O 2, 5 % CO 2 인큐베이터로 세포를 이동하고 신선한 쥐의 뇌 내피 세포 완전한 매체와 함께 제공하고, 37 ℃에서 1 시간 동안 배양한다. 참고 :이 단계는 재산 소화 상황을 나타냅니다. </li><li> 면역 현지화 및 로다 민 팔로이 딘 라벨링 챔버 슬라이드를 사용하여 (제 3 장 참조). </li></ol> 로다 민 Phalloidin의 사용 ZO-1과 F 액틴 라벨링 3. 면역 형광 현지화 <ol><li> 잘 1 시간 동안 OPTI-MEM / 감소 된 혈청 미디어 / 100 ㎕에 RBMEC의 단일 층을 포함하는 챔버 슬라이드를 노출. 인산염 완충 식염수 (PBS, pH를 7.0-7.2) 100 ㎕에 챔버 슬라이드 3 회 반복한다. </li><li> (PH 7.0-7.2)를 15 분 동안 PBS에 (PH 7.0-7.2)을 4 % 파라 포름 알데히드 100 μl를 사용하여 세포를 수정하고 PBS로 3 번 이상 챔버 슬라이드를 씻는다. </li><li> 또 다른 15 분 동안 PBS에서 0.5 % 트리톤 X-100 100 ㎕ (pH가 7.0-7.2)를 사용하여 세포를 Permeabilize 하시려면. 시간 동안 PBS에 2 % 소 혈청 알부민 (BSA) 100 ㎕로 차단. ZO-1 또는 F-액틴에 대해이 단계 중 하나를 얼룩 / 라벨 세포 중 하나 후. <ol><li> 면역 역을 위해150, 4 ℃에서 N / O 2 % BSA-PBS에서 제조 : ining 1에 ZO-1에 대하여 항 - 토끼 항체와 함께 일차 세포를 배양한다. , PBS로 세포에게 (PH 7.0-7.2)을 3 회 반복한다. 플루 오레 신 이소 티오 시아 네이트 (FITC) 100 ㎕와 함께 인큐베이션은 실온에서 1 시간 동안 항 - 토끼 이차 항체를 -tagged. </li><li> 로다 민 Phalloidin의 라벨링, 블로킹 다음, 20 분 동안 2 % BSA-PBS에서 제조 1:50 희석 로다 민 팔로이 딘 100 ㎕에 세포를 노출. </li><li> 면역 형광 염색과 PBS에서 로다 민 팔로이 딘 라벨, (PH 7.0-7.2)에서 세포를 씻으십시오. DAPI와 안티 - 페이드 시약을 포함하는 설치 미디어를 사용하여 챔버를 탑재합니다. </li></ol></li><li> 60 X 물 침지 렌즈를 사용하여 세포를 시각화하고 세포를 공 초점 현미경으로 단일 광학 평면에 스캔됩니다. </li></ol>

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The authors have nothing to disclose.

허혈 - 재관류 손상에 대한 생체 외 모델로 OGD-R은 잘 신경 세포 (10, 11)을 공부 설립되었습니다. 투과도 및 TJ 무결성 9 뇌 내피 세포 및 변경에 OGD의 효과를 나타내는 연구가있다. 그러나, 우리의 연구는 다음과 같은 허혈성 뇌졸중 발생 생체 조건에서 허혈성 재관류 손상에 가까운 표현이다 OGD의 효과뿐만 아니라 재산 소화를 보여줍니다. 저산소 ...

허혈 재관류 (IR)은 부상 뇌졸중, 심근 경색, 외상, 말초 혈관 질환, 외상성 뇌 손상과 관련된 다양한 1,2- 쇠약 합병증 및 사망의 원인으로 자주 발견된다. 뇌 혈관 IR 부상 염증과 부종 3에 이르는 초기 보조 부상입니다. 산화 및 염증 다음 대사성 스트레스의 결과로서 발생하는 합병증의 하나는 항상성 자유 라디칼 형성에 주요한 균형, 혈액 - 뇌 장벽의 변화 (BBB) ​​단단한 접합부 (TJS) 및 미세 혈관 투과성 4,5의 손실이다. 현재, BBB에 IR 손상의 효과를 연구하기 위해 사용되는 생체 내 모델에서 중간 대뇌 동맥 폐색 (MCAO) microembolism 및 트랜스 제닉 또는 녹아웃 동물을 포함한다. Hossmann 6 논의 그러나, 각각의 단점과 한계가있다. MCAO 모델은 effec을 연구하는 데 사용됩니다산화 환원 스트레스의 TS, BBB의 접합부 통신의 변화와 뇌와 면역 세포 사이의 상호 작용. 그러나, 이들은 그러한 내부에 정밀한 미세 수술 절차 및 어려움에 대한 요구와 같은 다양한 기술적 과제를 제시한다. 뇌허혈을 연구하는 유전자 변형 또는 녹아웃 동물의 사용이 경색 형성에 유전자에 의존하는 분자의 영향, 혈관 해부학의 변화와 다양한 체중 (6)과 같은 문제가있을 수 있습니다 동안 Microembolism는 순간적으로 BBB를 분해. 따라서, 허혈의 체외 모델은 의약품에 대한 역학적 연구를 수행하는 그들의 적용에 주로 최근에 관심을 증가 발견했다. 그러나, 시험관 연구의 결과는 완전히 생체 연구를 나타내지 있으므로 취급시주의를 6으로 해석해야합니다. 내피 세포의 단층 및 미세 혈관 투과성이 낮은 산소 농도의 반작용 효과가 있었다오가와 (7)에 의해 연구. 쥐의 뇌 미세 혈관 내피 세포 (RBMECs)는 체외 BBB을 개발하는데 사용되었다. 이 프로토콜에 제시된 산소 포도당 박탈과 재산 소화 (OGD-R) 기술은 Zulueta 등의 알과 주홍 등 8,9에 의해 연구에서 적용되고있다. 우리는 0 % O 2, 5 % CO 2 및 95 % N 2를 포함하는 저산소증 / 무산소 증 챔버에 넣어서 OGD-R에 뇌 내피 세포를 노출시켰다. 세포는 나중에 각각 면역 현지화 및 로다 민 팔로이 딘 라벨을 사용하여 TJ 무결성 및 스트레스 섬유 형성의 변화에​​ 대해 평가 하였다. zonula occludens-1의 (ZO-1)의 면역 형광 염색 ZO-1과 같이, TJ의 무결성을 판단하기 위해 수행된다 TJ 단백질 결합에 중요한 발판 막이다. 로다 민 Phalloidin의 라벨링은 세포 골격의 사상 액틴 수 (F- 액틴)를 결정하고, 내피 세포에서 액틴 스트레스 섬유 형성의 명확한 표시이다. &lt;P 클래스 = &quot;jove_content는&quot;&gt;이 방법의 목적은 BBB 내피 세포 TJ 무결성 및 F 액틴 스트레스 섬유 형성을 연구하기위한 생체 외 IR 모델로서 OGD-R의 개발에 대한 통찰력을 제공하는 것이다. 결과는 TJ 단백질의 운명에 대한 정보를 제공, ZO-1, R-OGD 다음 스트레스 섬유 형성한다. 이러한 관계를 이해하는 것은 OGD-R을 다음 트리거되는 기본 분자 메커니즘을 결정하고 OGD-R 처리 다음 BBB 중단을 향상시키기 위해 잠재적 인 치료 전략을 개발 할 수있는 기회를 제공 할 것입니다.

<table border="0" cellpadding="0" cellspacing="0" width="917">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Name of Material/ Equipment</td>
			<td style="width:167px;">Company</td>
			<td style="width:129px;">Catalog Number</td>
			<td style="width:251px;">Comments/Description</td>
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			<td height="21" style="height:21px;width:371px;">Proox model 110</td>
			<td style="width:167px;">Biospherix</td>
			<td style="width:129px;">Model 110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">DMEM, no glucose</td>
			<td style="width:167px;">Gibco, Life technologies</td>
			<td style="width:129px;">11966-025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Rhodamine Phalloidin</td>
			<td style="width:167px;">Life technologies</td>
			<td style="width:129px;">R415</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">ZO-1 Rabbit Polyclonal Antibody</td>
			<td style="width:167px;">Life technologies</td>
			<td align="right" style="width:129px;">617300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nunc Lab Tek II-CC 8&#160;well sterile, glass&#160;slides&#160;</td>
			<td style="width:167px;">Thermo scientific</td>
			<td align="right" style="width:129px;">177402</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">FITC-tagged anti-rabbit secondary antibody&#160;</td>
			<td style="width:167px;">Santa cruz</td>
			<td style="width:129px;">sc-2090</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:371px;">DPBS 1X</td>
			<td style="width:167px;">Thermo scientific</td>
			<td style="width:129px;">SH 30028.03</td>
			<td>Any other PBS available can be used</td>
		</tr>
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oxygen-glucose deprivation and reoxygenation as an in vitro ischemia-reperfusion injury model for studying blood-brain barrier dysfunction

허혈 재관류 (IR)는 부상 허혈성 뇌졸중과 관련된 이환율과 사망율에 크게 기여하는 것으로 알려져있다. 허혈성 뇌 혈관 사고는 모든 뇌졸중의 80 %를 차지한다. IR 부상의 일반적인 원인은 활성 산소의 형성을 유발 조직에 혈액, 영양소, 산소의 급성 / 만성 폐색 다음과 같은 유체의 급속한 유입이다. 허혈성 뇌졸중은 혈액 - 뇌 장벽 (BBB)​​ 부전 vasogenic 뇌부종 따른다. 구조적으로, 혈관 내피 세포 간의 긴밀한 접합 (TJS)은 혈액 - 뇌 장벽 (BBB)​​의 무결성을 유지하는데 중요한 역할을한다. IR 부상이 아닌 특정 염증 반응으로 이어지는 초기 보조 부상입니다. 염증 다음 산화 및 신진 대사에 영향을 미치는 스트레스는 BBB 투과성 꽉 접합 (TJ) 무결성의 중단을 포함하여 보조 뇌 손상을 트리거합니다. 우리의 프로토콜은 체외 제공 &lt;/ EM&gt; 쥐 뇌 내피 세포 TJ 무결성 및 스트레스 섬유 형성에 산소 - 글루코스 손실과 재산 소화 (OGD-R)의 예. 현재 생체 내 모델에서 여러 실험은 IR 손상의 효과를 연구하기 위해 사용된다; 그러나 그들은 이러한 기계적인 관계를 공부 수술, 유전자 따라 분자의 영향과 어려움을 수행함에있어 기술적 문제 등 여러 가지 한계를 가지고. 그러나, 체외 모델은 그 많은 한계를 극복하는 데 도움이 있습니다. 제시된 프로토콜은 잠재적 인 치료 전략을 제공하는 다양한 분자 메커니즘 및 기계적인 관계를 연구하기 위해 사용될 수있다. 그러나, 체외 연구 결과는 생체 내 연구에서 표준과 다를 수 있습니다주의를 해석되어야한다.

피브로넥틴 프리 코트 눈크 II 챔버 슬라이드에 배양 한 세포를 Biospherix ProOx 모델 (110)에 실 어서 OGD-R을 행 하였다. 로다 민 팔로이 딘 얼룩 라벨을 사용하여 F-액틴 스트레스 섬유 형성을 나타내는도 2와 골격 조립체에 도시 된 바와 같이도 3에 도시 된 바와 같이 OGD-R에 셀을 실시 후, 그들은 면역 형광 기술을 사용하여 ZO-1 접합부 염색을 위해 처리 하였다. 있었다 대조군 ?...

Watch this Scientific Journal Video about Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction at JoVE.com

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is associated with a high rate of morbidity and mortality. The goal of the in vitro model of oxygen-glucose deprivation and reoxygenation (OGD-R) described here is to assess the effects of ischemia reperfusion injury on a variety of cells, particularly in blood-brain barrier (BBB) endothelial cells.

같은 산소 포도당 박탈과 재산 소화<em&gt; 체외</em&gt; 허혈 - 재관류 손상 모델 공부 뇌 혈관 장벽 부전

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic ...

oxygen-glucose-deprivation-reoxygenation-as-an-vitro-ischemia

Research

JoVE Journal

Medicine

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

17.8K 조회수.  Texas A&M University Health Science Center College of Medicine. Ischemia-Reperfusion (IR) injury is associated with a high rate of morbidity and mortality. The goal of the in vitro model of oxygen-glucose deprivation and reoxygenation (OGD-R) described here is to assess the effects of ischemia reperfusion injury on a variety of cells, particularly in blood-brain barrier (BBB) endothelial cells. 

비디오: Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Normothermic Cardiac Arrest and Cardiopulmonary Resuscitation: A Mouse Model of Ischemia-Reperfusion Injury

Acute Kidney Injury (AKI) is a common, highly lethal, complication of critical illness which has a high mortality1-4 and which is most 
frequently caused by whole-body hypoperfusion.5,6 Successful reproduction of whole-body hypoperfusion in rodent models has been fraught with 
difficulty.7-9,9,10 Models which employ focal ischemia have repeatedly demonstrated results which do not translate to the clinical setting, and larger 
animal models which allow for whole body hypoperfusion lack access to the full toolset of genetic manipulation possible in the mouse.11,12 However, in recent 
years a mouse model of cardiac arrest and cardiopulmonary resuscitation has emerged which can be adapted to model AKI.13 This model reliably reproduces 
physiologic, functional, anatomic, and histologic outcomes seen in clinical AKI, is rapidly repeatable, and offers all of the significant advantages of a murine surgical 
model, including access to genetic manipulative techniques, low cost relative to large animals, and ease of use. Our group has developed extensive experience 
with use of this model to assess a number of organ-specific outcomes in AKI.14,15

A powerful model for perioperative and critical care related acute kidney injury is presented. Using whole body hypoperfusion induced by cardiac arrest it is possible to nearly replicate the histologic and functional changes of clinical AKI.

Normothermic 심장 마비와 심폐 소생술의 : 국소 빈혈 - Reperfusion 상해의 마우스 모델

Acute Kidney Injury (AKI) is a common, highly lethal, complication of critical illness which has a high mortality1-4 and which is most 
frequently caused ...

연구

의학

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and finding new sources for implantable cells are the focus of recent research4,5. Investigating the effectiveness of cell therapies is not an easy task and new tools are needed to investigate the mechanisms involved in the treatment process6. We designed an experimental protocol of ischemia/reperfusion in order to allow the observation of cellular connections and even subcellular mechanisms during ischemia/reperfusion injury and after stem cell transplantation and to evaluate the efficacy of cell therapy. H9c2 cardiomyoblast cells were placed onto cell culture plates7,8. Ischemia was simulated with 150 minutes in a glucose free medium with oxygen level below 0.5%. Then, normal media and oxygen levels were reintroduced to simulate reperfusion. After oxygen glucose deprivation, the damaged cells were treated with transplantation of labeled human bone marrow derived mesenchymal stem cells by adding them to the culture. Mesenchymal stem cells are preferred in clinical trials because they are easily accessible with minimal invasive surgery, easily expandable and autologous. After 24 hours of co-cultivation, cells were stained with calcein and ethidium-homodimer to differentiate between live and dead cells. This setup allowed us to investigate the intercellular connections using confocal fluorescent microscopy and to quantify the survival rate of postischemic cells by flow cytometry. Confocal microscopy showed the interactions of the two cell populations such as cell fusion and formation of intercellular nanotubes. Flow cytometry analysis revealed 3 clusters of damaged cells which can be plotted on a graph and analyzed statistically. These populations can be investigated separately and conclusions can be drawn on these data on the effectiveness of the simulated therapeutical approach.

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

We demonstrate how to set up an in vitro ischemia/reperfusion model and how to evaluate the effect of stem cell therapy on postischemic cardiac cells.

에서 간세포 이식 체외에서 시뮬레이션 국소 빈혈 / Reperfusion 모델

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

Ischemia-reperfusion Model of Acute Kidney Injury and Post Injury Fibrosis in Mice

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often unreported mortality rates that may confound analyses. Bilateral renal pedicle clamping is commonly used to induce IR-AKI, but differences between effective clamp pressures and/or renal responses to ischemia between kidneys often lead to more variable results. In addition, shorter clamp times are known to induce more variable tubular injury, and while mice undergoing bilateral injury with longer clamp times develop more consistent tubular injury, they often die within the first 3 days after injury due to severe renal insufficiency. To improve post-injury survival and obtain more consistent and predictable results, we have developed two models of unilateral ischemia-reperfusion injury followed by contralateral nephrectomy. Both surgeries are performed using a dorsal approach, reducing surgical stress resulting from ventral laparotomy, commonly used for mouse IR-AKI surgeries. For induction of moderate injury BALB/c mice undergo unilateral clamping of the renal pedicle for 26 min and also undergo simultaneous contralateral nephrectomy. Using this approach, 50-60% of mice develop moderate AKI 24 hr after injury but 90-100% of mice survive. To induce more severe AKI, BALB/c mice undergo renal pedicle clamping for 30 min followed by contralateral nephrectomy 8 days after injury. This allows functional assessment of renal recovery after injury with 90-100% survival. Early post-injury tubular damage as well as post injury fibrosis are highly consistent using this model.

We describe models of moderate and severe ischemia-reperfusion-induced kidney injury in which the mice undergo unilateral renal pedicle clamping followed by simultaneous or delayed contralateral nephrectomy, respectively. These models consistently give rise to renal dysfunction and post-injury fibrosis, but injury severity and survival are dependent on mouse background, age and surgical equipment.

마우스의 급성 신장 손상 및 사후 상해 섬유증의 허혈 - 재관류 모델

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often ...

Stretch in Brain Microvascular Endothelial Cells (cEND) as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying mechanisms involved in it are useful for treatment. There are both in vivo and in vitro methods available for this purpose. In vivo models can mimic actual head injury as it occurs during TBI. However, in vivo techniques may not be exploited for studies at the cell physiology level. Hence, in vitro methods are more advantageous for this purpose since they provide easier access to the cells and the extracellular environment for manipulation.
Our protocol presents an in vitro model of TBI using stretch injury in brain microvascular endothelial cells. It utilizes pressure applied to the cells cultured in flexible-bottomed wells. The pressure applied may easily be controlled and can produce injury that ranges from low to severe. The murine brain microvascular endothelial cells (cEND) generated in our laboratory is a well-suited model for the blood brain barrier (BBB) thus providing an advantage to other systems that employ a similar technique. In addition, due to the simplicity of the method, experimental set-ups are easily duplicated. Thus, this model can be used in studying the cellular and molecular mechanisms involved in TBI at the BBB.

Stretch in Brain Microvascular Endothelial Cells cEND as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

In vitro traumatic brain injury models are being developed to reproduce in vivo brain deformation. Stretch-induced injury has been employed for astrocytes, neurons, glial cells, aortic, and brain endothelial cells. However, our system uses a blood brain barrier (BBB) model that possesses properties constituting a legitimate model of the BBB to establish an in vitro&#160;TBI model.

로 뇌 미세 혈관 내피 세포 (CEnd를)에 기지개<em&gt; 체외</em혈액 뇌 장벽의&gt; 외상성 뇌 손상 모델

Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying ...

Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm&#183;cm2 on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 &#177;&#160;0.11 x 10-3 cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.

Setting-up an In Vitro Model of Rat Blood-brain Barrier BBB: A Focus on BBB Impermeability and Receptor-mediated Transport

The aim of the present study was to validate the reproducibility of an in vitro BBB model involving a rat syngeneic co-culture of endothelial cells and astrocytes. The endothelial cell monolayer presented high TEER and low LY permeability. Expression of specific TJ proteins, functional responses to inflammation and functionality of transporters and receptors were assessed.

설정 - 업<em&gt; 체외</em쥐 뇌 혈관 벽의&gt; 모델 (BBB)​​ : BBB 불 침투성 및 수용체 매개 운송에 초점

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and ...

Murine Model of Intestinal Ischemia-reperfusion Injury

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, hypotension, necrotizing enterocolitis, bowel transplantation, trauma and chronic inflammation. Intestinal ischemia-reperfusion (IR) injury is a consequence of acute mesenteric ischemia, caused by inadequate blood flow through the mesenteric vessels, resulting in intestinal damage. Reperfusion following ischemia can further exacerbate damage of the intestine. The mechanisms of IR injury are complex and poorly understood. Therefore, experimental small animal models are critical for understanding the pathophysiology of IR injury and the development of novel therapies.
Here we describe a mouse model of acute intestinal IR injury that provides reproducible injury of the small intestine without mortality. This is achieved by inducing ischemia in the region of the distal ileum by temporally occluding the peripheral and terminal collateral branches of the superior mesenteric artery for 60 min using microvascular clips. Reperfusion for 1 hr, or 2 hr after injury results in reproducible injury of the intestine examined by histological analysis. Proper position of the microvascular clips is critical for the procedure. Therefore the video clip provides a detailed visual step-by-step description of this technique. This model of intestinal IR injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Here we describe the detailed procedure of intestinal ischemia-reperfusion in mice which results in reproducible injury without mortality to encourage the standardization of this technique across the field. This model of intestinal ischemia-reperfusion injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

창자 허혈 - 재관류 손상의 쥐 모델

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

생체 외에서 혈액-뇌 장벽 돼지 뇌 내 피 세포를 기반으로 하는 모델의 설립에 대 한 방법 개선

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

Evaluation of Coronary Flow Reserve After Myocardial Ischemia Reperfusion in Rats

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via recanalization of the coronary artery is the most effective strategy for reducing the size of ischemic myocardium. The coronary microvasculature cannot be visualized and imaged&#160;in vivo, but there are several invasive and noninvasive techniques that can be used to assess parameters which depend directly on coronary microvascular function. The endothelial function after ischemia reperfusion can be assessed also at the level of the coronary circulation via the coronary flow reserve (CFR).&#160;In this study, peak velocity of left anterior descending (LAD) coronary arteries was measured in rats in vivo via Transthoracic Doppler Echocardiography during resting and stress challenge (induced by Dobutamine). A normal heart can increase its coronary blood flow up to four times above the resting values during stress induction. Following ischemia reperfusion, we found a significantly diminished CFR, which can be used as a marker of coronary microvascular dysfunction. CFR has opened a window on the importance of microvascular dysfunction and has been shown to predict cardiovascular risk independent of whether the severe obstructive disease is present.

Coronary flow reserve (CFR), is defined as the ratio of maximal coronary blood flow to the resting coronary blood flow. We present a protocol for evaluating CFR in rats via ultrasound, which offers the opportunity to predict cardiovascular risk factors in the absence of obstructive coronary disease.

쥐의 심근 허혈 재관류 후 관상 동맥 흐름 예비의 평가

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via ...

A Pre-clinical Rat Model for the Study of Ischemia-reperfusion Injury in Reconstructive Microsurgery

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many areas of biomedical research due to its cost-effectiveness and its translation to humans. This protocol describes a method to create a preclinical free skin flap model in rats with ischemia-reperfusion injury. The described 3 cm x 6 cm rat free skin flap model is easily obtained after the placement of several vascular ligatures and the section of the vascular pedicle. Then, 8 h after the ischemic insult and completion of the microsurgical anastomosis, the free skin flap develops the tissue damage. These ischemia-reperfusion injury-related damages can be studied in this model, making it a suitable model for evaluating therapeutic agents to address this pathophysiological process. Furthermore, two main monitoring techniques are described in the protocol for the assessment of this animal model: transit-time ultrasound technology and laser speckle contrast analysis.

Here, we describe a pre-clinical animal model for studying the pathophysiology of ischemia-reperfusion injury in reconstructive microsurgery. This free skin flap model based on the superficial caudal epigastric vessels in the rat may also allow for the evaluation of different therapies and compounds to counteract ischemia-reperfusion injury-related damage.

재건 미세 수술에서 허혈 재관류 손상의 연구를위한 전 임상 쥐 모델

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many ...

Delayed Intramyocardial Delivery of Stem Cells after Ischemia Reperfusion Injury in a Murine Model

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, cardiac stem cell therapy is studied by delivering SCs concurrently with the induction of myocardial injury. However, this approach presents two significant limitations: the early hostile pro-inflammatory ischemic environment may affect the survival of transplanted SCs, and it does not represent the subacute infarction scenario where SCs will likely be used. Here we describe a two-part series of surgical procedures for the induction of ischemia-reperfusion injury and delivery of mesenchymal stem cells (MSCs). This method of stem cell administration may allow for the longer viability and retention around damaged tissue by circumventing the initial immune response. A model of ischemia reperfusion injury was induced in mice accompanied by the delivery of mesenchymal stem cells (3.0 x 105), stably expressing the reporter gene firefly luciferase under the constitutively expressed CMV promoter, intramyocardially 7 days later. The animals were imaged via ultrasound and bioluminescent imaging for confirmation of injury and injection of cells, respectively. Importantly, there was no added complication rate when performing this two-procedure approach for SC delivery. This method of stem cell administration, collectively with the utilization of state-of-the-art reporter genes, may allow for the in vivo study of viability and retention of transplanted SCs in a situation of chronic ischemia commonly seen clinically, while also circumventing the initial pro-inflammatory response. In summary, we established a protocol for the delayed delivery of stem cells into the myocardium, which can be used as a potential new approach in promoting regeneration of the damaged tissue.

Stems cells are continuously investigated as potential treatments for individuals with myocardial damage, however, their decreased viability and retention within injured tissue can impact their long-term efficacy. In this manuscript we describe an alternative method for stem cell delivery in a murine model of ischemia reperfusion injury.

뮤린 모델에서 허혈혈 재관류 부상 후 줄기 세포의 지연 된 자궁 내 납품

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, ...