我们承认斯科特和怀特医院研究资助计划提供财政支持和得克萨斯州A＆M大学健康科学中心大学的医学影像学综合实验室，利用激光共聚焦显微镜。我们承认格伦克莱尔先生的帮助，稿件编辑。

1.播种内皮细胞<ol><li>从成年SD大鼠获得RBMEC的原代培养（或获得这些商业）。 </li><li>培育在百厘米纤连蛋白（50微克/毫升）使用大鼠脑内皮细胞生长介质涂覆培养皿RBMECs。改变介质每两天，直到汇合为止。 </li><li>在达到80-90％汇合，轻轻涡旋洗细胞在5ml磷酸盐缓冲盐水（PBS）中。然后将细胞通过暴露至1毫升温的0.25％胰蛋白酶乙二胺四乙酸（EDTA）溶液，平衡至37℃的脱离。 </li><li>孵育细胞在37℃下2-5分钟，直至细胞被分离并分散。 注:点击培养皿分离细胞。在显微镜下检视细胞以确认细胞完全脱离从盘的表面上。 </li><li>加入5 ml完全培养基的培养皿中，以中和胰蛋白酶。吸取出包含分离的细胞的培养基中，并收集成一个15毫升的离心管中。 。 </li><li>离心含有内皮细胞，在220×g离心5分钟介质。 </li><li>吸出上清液并保留沉淀含有cells.Suspend沉淀成3-5毫升新鲜大鼠脑内皮细胞生长培养基轻轻混合，上下用吸管将细胞将被随后使用自动细胞计数器或血细胞计数器计数。 </li><li>用的接种密度，每孔10,000-15,000细胞之间的范围内细胞悬液转移入纤维连接蛋白（50微克/毫升）预涂8孔无菌室载玻片系统，0.7 平方厘米/孔。生长的细胞在37℃，直至汇合达到 注意:用于执行蛋白质印迹或其他实验中，细胞可于10cm细胞培养皿或特别的菜肴所要求的实验生长。 </li></ol> 2.氧气和葡萄糖剥夺复氧体外模型注:以下协议已被改编自苏卢埃塔等 。 1997;朱H 等 2012 8,9。 <ol><li>使用低氧细胞培养系统来研究RBMECs OGD-R的中的效果（ 见图1）。设置和校准的缺氧细胞培养系统在开始实验之前，根据制造商的说明。 </li><li>从37℃培养箱中取出汇合玻片（步骤1.8）。替换在用脱氧腔滑动的完全培养基，无葡萄糖，Dulbecco氏改良的Eagle氏培养基（DMEM）中，并置于低氧室95％以下，N 2和5％CO 2下2小时，在37℃，以代表OGD条件。 </li><li>移动单元返回到培养箱中95％ 的 O 2，5％的CO 2在37℃，并提供新鲜的大鼠脑的内皮细胞完全培养基并孵育另外1小时，在37℃。 注:此步骤表示复氧的局面。 </li><li>使用玻片进行免疫荧光定位和鬼笔罗丹明标记（见第3节）。 </li></ol>同时ZO-1 和 F肌动蛋白标签使用罗丹明鬼笔环肽免疫3.本地化<ol><li>揭露含有RBMEC单层至100μlOPTI-MEM /降低血清培养基/孔的1小时的玻片。在100μl磷酸盐缓冲盐水（PBS，pH值为7.0-7.2）的洗室载玻片3次。 </li><li>固定用100μl的4％多聚甲醛的PBS（pH值7.0-7.2）的细胞15分钟，并在PBS中洗室玻片3次以上（pH值为7.0-7.2）。 </li><li>透用100微升0.5％的Triton X-100的PBS，（pH值7.0-7.2）另外15分钟的细胞。用100μl的2％牛血清白蛋白（BSA）的PBS一小时阻塞。在此步骤之后或者染色/标记的细胞或者为ZO-1或F-肌动蛋白。 <ol><li>对于免疫STA进不去孵育将细胞与抗ZO-1中1的抗兔一抗:150，用于O-制备2％BSA-PBS / N在4℃。在PBS中洗涤细胞3次，（pH值7.0-7.2）。孵化用100μl异硫氰酸荧光素（FITC）的-tagged抗兔二级抗体进行1小时，在室温。 </li><li>为若丹明鬼笔环肽标记，以下阻挡，暴露细胞至100μl罗丹明鬼笔环肽的1:50稀释，在2％BSA-PBS制备，20分钟。 </li><li>从免疫荧光染色和若丹明鬼笔环肽标记在PBS，（pH值7.0-7.2）洗细胞。安装使用含抗褪色试剂用DAPI安装媒体室。 </li></ol></li><li>使用60×水浸没透镜可视化的细胞和细胞共聚焦显微镜下扫描在一个单一的光学平面。 </li></ol>

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The authors have nothing to disclose.

OGD-R作为体外模型缺血再灌注损伤已经确立为研究神经元10,11。也有研究显示，OGD对脑血管内皮细胞和变更在渗透性和TJ完整性9的效果。然而，我们的研究显示，OGD的效果以及复氧，它是缺血再灌注损伤的发生的缺血性中风的体内条件下仔细表示。 缺氧缺血性条件是已知的诱发炎症在中枢神经系统中，导致通过增加细胞旁渗透性和血管性水肿4至BBB破坏。再灌注损伤是一个非常复杂的，动态的过程，其中有过量的活性氧的产生，ATP耗竭，上升外钾，兴奋性神经递质，内皮细胞和神经细胞肿胀，免疫细胞活化的释放。这反过来Çauses激活各种炎症途径，导致细胞因子的产生，诱导核酸酶和蛋白酶，导致水肿，所有这些都显示出激活各种半胱氨酸蛋白酶，导致血脑屏障完整性5,12中断。内皮细胞是特别容易出现缺血性损伤，由于大量线粒体的存在。相邻的内皮细胞是由通过紧密连接蛋白保持紧密连接而彼此连接。 ZO-1被示为在与其他紧密连接蛋白保持TJ完整性通过其相互作用中发挥重要作用，并&#160;肌动蛋白细胞骨架组装。此外，肌动蛋白细胞骨架的精确调节是许多发育和生理过程，包括细胞 - 细胞粘连是必不可少的。在内皮细胞，肌动蛋白应力纤维形成被认为是与屏障功能障碍和高通透性13,14相关联。罗丹明phalloi嚣在本研究中用于观察F-肌动蛋白应力纤维形成标记技术是终点研究。然而，也可以进行的应力纤维形成动态和实时成像如图格特和布雷斯林15。 目前的研究majorly强调ZO-1的对BBB紧密连接的完整性的贡献。然而，其他的紧密连接分子如紧密连接蛋白5，紧密连接，连接粘附分子（JAM）等也可以被用作标记，以研究以下OGD-R的所述BBB紧密连接的完整性。在这项研究中，我们采用免疫荧光定位和F-肌动蛋白标记作为定性的技术来确定紧密连接的完整性。然而，我们也可以研究血脑屏障的使用荧光标记物和跨内皮电阻（TEER）的另一重要特征像渗透性的定量测量。 使用该OGD-R技术呈现在这项研究中Biospherix系统只能被用于在氧的固定浓度缺氧/缺氧研究;然而，我们不能使用该系统用于研究不同氧浓度对内皮细胞的作用。为了研究不同浓度的氧对内皮细胞的影响，我们需要采用适合于目的的其他可用的模型。 这种技术可以用于研究调节血脑屏障渗透性过高和TJ完整性各种细胞和分子事件之间的机械关系。了解他们将为制定各种分子策略，以减轻BBB破坏和微血管通透性的洞察力。

缺血-再灌注（IR）的损伤被发现是与中风，心肌梗塞，外伤，周围血管疾病和外伤性脑损伤1,2-相关的各种衰弱的并发症和死亡的常见原因。 IR损伤的脑血管是早期的继发性损伤，导致炎症和水肿3。之一的严重并发症发生的氧化和代谢应激以下炎症的结果是自我平衡，导致自由基的形成，在血脑屏障改变（BBB）的紧密连接（紧密连接）和微血管通透性4,5的损失。 目前，用于研究在BBB缺血再灌注损伤的影响体内模型包括大脑中动脉闭塞（缺血），微栓塞，以及转基因或基因敲除动物。然而，每一个都有它的缺点和局限性通过Hossmann 6所讨论的。 MCAO模型被用于研究短跑运动员氧化还原应力的TS，在血脑屏障的交界通信和改变脑和免疫细胞之间的相互作用。然而，它们存在各种技术的挑战，如需要精确显微手术操作和困难在其中。瞬间微栓塞打破了BBB，而利用转基因或基因敲除动物的研究脑缺血可能具有类似基因分子依赖的影响，形成梗塞，改变血管解剖和不同体重6的挑战。因此，缺血的体外模型发现增加近倍，主要由于息其适用性在执行机理研究的药物。但是， 在体外研究的结果可能不完全代表在体内研究，必须谨慎6进行解释。 对血管内皮细胞单层和微血管通透性低氧气浓度的反作用已研究了小川7。大鼠脑微血管内皮细胞（RBMECs）用于开发体外 BBB。氧糖剥夺复氧（OGD-R）技术，该协议提出了改编自苏卢埃塔等人 ，朱等人的研究8,9。我们暴露脑内皮细胞对OGD-R的通过将它们放置在含有0％ 的 O 2，5％CO 2和95％N 2和缺氧/缺氧室中。细胞后评估分别采用免疫荧光定位和罗丹明鬼笔标记在TJ的完整性和应力纤维形成的改变。被执行以确定TJ完整性免疫荧光染色为紧密连接-1（ZO-1），如ZO-1是一种重要的脚手架膜结合的TJ蛋白。罗丹明鬼笔环肽标记决定了肌动蛋白丝状（F肌动蛋白）的细胞骨架，是肌动蛋白应力纤维形成的血管内皮细胞的明确指示。 &lt;P类="jove_content"&gt;该方法的目标是提供洞察显影OGD-R作为体外的IR模型用于研究血脑屏障内皮细胞的TJ完整性和F-肌动蛋白应力纤维形成。研究结果将提供TJ蛋白的命运的消息，ZO-1和应力纤维形成以下OGD-R。了解这些关系将提供一个机会，以确定被触发以下OGD-R潜在的分子机制，并开发潜在的治疗策略，以提高血脑屏障破坏OGD如下-R处理。

<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<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>
		</tr>
		<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>
	</tbody>
</table>

oxygen-glucose deprivation and reoxygenation as an in vitro ischemia-reperfusion injury model for studying blood-brain barrier dysfunction

缺血 - 再灌注（IR）的损伤是已知显著与缺血性中风的发病率和死亡率。缺血性脑血管意外占80％的中风。 IR损伤的常见原因是流体的以下血液，营养物，氧的急性/慢性闭塞于组织引发的自由基的形成的快速流入。 缺血性中风之后是血 - 脑屏障（BBB）的功能障碍和血管源性脑水肿。在结构上，所述的内皮细胞之间的紧密连接（紧密连接）在保持血 - 脑屏障（BBB）的完整性方面发挥重要作用。 IR损伤是早期继发性损伤导致的非特异性，炎症反应。氧化和代谢应激下炎症引发的继发性脑损害，包括血脑屏障通透性及紧密连接（TJ）的完整性的破坏。 我们的协议提出了一种在体外&lt;/ em&gt;的例子对大鼠脑内皮细胞TJ的完整性和应力纤维形成缺氧缺糖复氧（OGD-R）。目前，几个实验的体内模型用于研究IR损伤的影响;然而，它们具有一些局限性，如在执行手术，基因依赖分子影响和困难在研究机械关系的技术挑战。然而， 在体外模型可能有助于克服许多这些限制。所提出的协议可用于研究各种分子机制和机理的关系，以提供潜在的治疗策略。但是， 在体外研究的结果可以不同于标准体内研究和应谨慎解释。

细胞纤连蛋白预涂Nunc公司II腔室玻片中培养通过放置在一个Biospherix ProOx模型110室中进行OGD-R上。使细胞对OGD-R的后，将它们控制的细 ​​胞是处理用于使用免疫荧光技术ZO-1交界染色， 如图2和细胞骨架组装表示使用若丹明鬼笔环肽染色标签F-肌动蛋白应力纤维形成， 如图3。未进行OGD-R的显示持续交界的完整性，同时进行OGD-R的内皮细胞表现出不连续的路口，指示TJ完整性图2的损失。即不进行OGD-R的处理的对照细胞显示没有或最小˚F肌动蛋白应力纤维形成，同时进行OGD-R的内皮细胞表现出增加的˚F肌动蛋白应力纤维形成指示的变化，肌动蛋白细胞骨架组装图3;众议员<html> RINT与许可参考＃13 <img alt="图1" src="/files/ftp_upload/52699/52699fig1.jpg" /> 图中的缺氧系统ProOx 110型1.典型配置 。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52536/52536fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52699/52699fig2.jpg" /> 紧密连接蛋白图2.免疫本地化（TJP）ZO-1以下的示范OGD-R在RBMECs紧密连接的破坏，如白色箭头。比例尺= 10微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52536/52536fig2large.jpg" target="_blank">请点击此处查看本一个更大的版本图。</a> "&gt; <img alt="图3" src="/files/ftp_upload/52699/52699fig3.jpg" /> 图3.若丹明鬼笔环肽标签适用于 F肌动蛋白纤维应力形成示范变化大会细胞骨架OGD之后-R在RBMECs，如白色箭头。比例尺= 10微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52536/52536fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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.

缺氧缺糖再给氧作为<em&gt;在体外</em&gt;缺血再灌注损伤模型的研究血脑屏障功能障碍

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

Nanyang Technological University

Research

JoVE Journal

Medicine

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

17.9K 次观看.  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. 

视频: 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.

常温心脏骤停和心肺复苏术:缺血再灌注损伤的小鼠模型

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 ...

科研

医学

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.

在干细胞移植在体外模拟缺血/再灌注模型

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.

急性肾损伤和伤后纤维化小鼠缺血再灌注模型

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.

作为脑微血管内皮细胞（CEND）的伸展<em&gt;体外</em创伤性脑损伤模型血脑屏障

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

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.

肠缺血再灌注损伤的小鼠模型

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.

一种基于猪脑内皮细胞的体外血脑屏障模型的改进方法

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.

大鼠心肌缺血再灌注后冠状动脉流动储备评价

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.

重建显微外科缺血再灌注损伤研究的临床前大鼠模型

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

Organ Ischemia-Reperfusion Injury by Simulating Hemodynamic Changes in Rat Liver Transplant Model

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, including ischemia-reperfusion injury (IRI) of extrahepatic organs. This model requires numerous experiments and devices. The duration of anhepatic phase is closely related to the time to develop IRI after transplantation. In this experiment, we used hemodynamic changes to induce extrahepatic organ damage in rats and determined the maximum tolerance time. The time until the most severe organ injury varied for different organs. This method can easily be replicated and can also be used to study IRI of the extrahepatic organs after liver transplantation.

This paper provides a detailed description of how to build an animal model of the anhepatic phase (liver ischemia) in rats to facilitate basic research into ischemia-reperfusion injury after liver transplantation.

器官缺血-输液损伤模拟大鼠肝脏移植模型的血动力学变化

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, ...

A Rat Lung Transplantation Model of Warm Ischemia/Reperfusion Injury: Optimizations to Improve Outcomes

From our experience with rat lung transplantation, we have found several areas for improvement. Information in the existing literature regarding methods for choosing appropriate cuff sizes for the pulmonary vein (PV), pulmonary artery (PA), or bronchus (Br) are varied, thus making the determination of proper cuff size during rat lung transplantation an exercise of trial and error. By standardizing the cuffing technique to use the smallest effective cuff appropriate for the size of the vessel or bronchus, one can make the transplantation procedure safer, faster, and more successful. Since diameters of the PV, PA, and Br are related to the body weight of the rat, we present a strategy to choosing an appropriate size using a weight-based guide. Since lung volume is also related to body weight, we recommend that this relationship should also be considered when choosing the proper volume of air for donor lung inflation during warm ischemia as well as for the proper volume of PBS to be instilled during bronchoalveolar lavage (BAL) fluid collection. We also describe methods for 4th intercostal space dissection, wound closure, and sample collection from both the native and transplanted lobes.

Here, we present optimizations to a rat lung transplantation model that serve to improve outcomes. We provide a size guide for cuffs based on body weight, a measurement strategy to ascertain the 4th intercostal space, and methods of wound closure and BAL (bronchoalveolar lavage) fluid and tissue collection.

温缺血/再灌注损伤的大鼠肺移植模型:改善结局的优化

From our experience with rat lung transplantation, we have found several areas for improvement. Information in the existing literature regarding methods ...