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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>

<ol><li>Eltzschig, H. K., Eckle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ischemia+and+reperfusion--from+mechanism+to+translation%5BTitle%5D)+AND+%22Nat+Med%22%5BJournal%5D)">Ischemia and reperfusion--from mechanism to translation.</a> Nat Med. 17 (11), 1391-1401 (2011).</li>
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<li>Zulueta, J. J., Sawhney, R., Yu, F. S., Cote, C. C., Hassoun, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intracellular+generation+of+reactive+oxygen+species+in+endothelial+cellsexposed+to+anoxia-reoxygenation%5BTitle%5D)+AND+%22Am+J+Physiol%22%5BJournal%5D)">Intracellular generation of reactive oxygen species in endothelial cellsexposed to anoxia-reoxygenation.</a> Am J Physiol. 272 (5 Pt 1), L897-L902 (1997).</li>
<li>Zhu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Baicalin+reduces+the+permeability+of+the+blood-brain+barrier+during+hypoxia+in+vitro+by+increasing+the+expression+of+tight+junction+proteins+in+brain+microvascular+endothelial+cells%5BTitle%5D)+AND+%22J+Ethnopharmacol%22%5BJournal%5D)">Baicalin reduces the permeability of the blood-brain barrier during hypoxia in vitro by increasing the expression of tight junction proteins in brain microvascular endothelial cells.</a> J Ethnopharmacol. 141 (2), 714-720 (2012).</li>
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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...

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><tbody><tr><td>Proox model 110</td><td>Biospherix</td><td>Model 110</td><td></td></tr><tr><td>DMEM, no glucose</td><td>Gibco, Life technologies</td><td>11966-025</td><td></td></tr><tr><td>Rhodamine Phalloidin</td><td>Life technologies</td><td>R415</td><td></td></tr><tr><td>ZO-1 Rabbit Polyclonal Antibody</td><td>Life technologies</td><td>617300</td><td></td></tr><tr><td>Nunc Lab Tek II-CC 8&amp;nbsp;well sterile, glass&amp;nbsp;slides&amp;nbsp;</td><td>Thermo scientific</td><td>177402</td><td></td></tr><tr><td>FITC-tagged anti-rabbit secondary antibody&amp;nbsp;</td><td>Santa cruz</td><td>sc-2090</td><td></td></tr><tr><td>DPBS 1X</td><td>Thermo scientific</td><td>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<s...

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

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

2-Vessel Occlusion/Hypotension: A Rat Model of Global Brain Ischemia

Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ischemia produces damage to specific brain regions shown to be highly sensitive to ischemia 1. Hippocampal neurons have higher sensitivity to ischemic insults compared to other cell populations, and specifically, the CA1 region of the hippocampus is particularly vulnerable to ischemia/reperfusion 2.
The design of therapeutic interventions, or study of mechanisms involved in cerebral damage, requires a model that produces damage similar to the clinical condition and in a reproducible manner. Bilateral carotid vessel occlusion with hypotension (2VOH) is a model that produces reversible forebrain ischemia, emulating the cerebral events that can occur during cardiac arrest and resuscitation. We describe a model modified from Smith et al. (1984) 2, as first presented in its current form in Sanderson, et al. (2008) 3, which produces reproducible injury to selectively vulnerable brain regions 3-6. The reliability of this model is dictated by precise control of systemic blood pressure during applied hypotension, the duration of ischemia, close temperature control, a specific anesthesia regimen, and diligent post-operative care. An 8-minute ischemic insult produces cell death of CA1 hippocampal neurons that progresses over the course of 6 to 24 hr of reperfusion, while less vulnerable brain regions are spared. This progressive cell death is easily quantified after 7-14 days of reperfusion, as a near complete loss of CA1 neurons is evident at this time.
In addition to this brain injury model, we present a method for CA1 damage quantification using a simple, yet thorough, methodology. Importantly, quantification can be accomplished using a simple camera-mounted microscope, and a free ImageJ (NIH) software plugin, obviating the need for cost-prohibitive stereology software programs and a motorized microscopic stage for damage assessment.

Bilateral carotid occlusion coupled with systemic hypotension produces global brain ischemia in the rat, resulting in damage to the hippocampus with reproducible severity. Animal subjects are impaired with predictable patterns of brain damage, they recover expediently, and mortality rates are comparatively low.

2血管闭塞/低血压：全脑缺血模型大鼠

Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ...

科研

医学

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.

设置了一个<em&gt;体外</em大鼠血脑屏障&gt;模型（BBB）：在BBB抗渗性和受体介导的运输为中心

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

The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional deficits in the affected patients. Despite intensive research efforts, there is still no effective treatment option available that reduces neuronal injury and protects neurons in the ischemic areas from delayed secondary death. Research in this area typically involves the use of elaborate and problematic animal models. Entorhino-hippocampal organotypic slice cultures challenged with oxygen and glucose deprivation (OGD) are established in&#160;vitro models which mimic cerebral ischemia. The novel aspect of this study is that changes of the brain blood vessels are studied in addition to neuronal changes and the reaction of both the neuronal compartment and the vascular compartment can be compared and correlated. The methods presented in this protocol substantially broaden the potential applications of the organotypic slice culture approach. The induction of OGD or hypoxia alone can be applied by rather simple means in organotypic slice cultures and leads to reliable and reproducible damage in the neural tissue. This is in stark contrast to the complicated and problematic animal experiments inducing stroke and ischemia in vivo. By broadening the analysis to include the study of the reaction of the vasculature could provide new ways on how to preserve and restore brain functions. The slice culture approach presented here might develop into an attractive and important tool for the study of ischemic brain injury and might be useful for testing potential therapeutic measures aimed at neuroprotection.

A protocol for entorhino-hippocampal organotypic slice cultures, which allows reproducing many aspects of ischemic brain injury, is presented. By studying changes of the neurovasculature in addition to changes in the neurons, this protocol is a versatile tool to study plastic changes in neural tissue after injury.

神经血管重塑的分析Entorhino，海马脑切片培养

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional ...

神经科学

A Triple Primary Cell Culture Model of the Human Blood-Brain Barrier for Studying Ischemic Stroke In Vitro

Ischemic stroke is a major cause of death and disability worldwide with limited therapeutic options. The neuropathology of ischemic stroke is characterized by an interruption in blood supply to the brain leading to cell death and cognitive dysfunction. During and after ischemic stroke, blood-brain barrier (BBB) dysfunction facilitates injury progression and contributes to poor patient recovery. Current BBB models primarily include endothelial monocultures and double co-cultures with either astrocytes or pericytes.
Such models lack the ability to fully imitate a dynamic brain microenvironment, which is essential for cell-to-cell communication. Additionally, commonly used BBB models often contain immortalized human endothelial cells or animal-derived (rodent, porcine, or bovine) cell cultures that pose translational limitations. This paper describes a novel well-insert-based BBB model containing only primary human cells (brain microvascular endothelial cells, astrocytes, and brain vascular pericytes) enabling the investigation of ischemic brain injury in vitro. 
The effects of oxygen-glucose deprivation (OGD) on barrier integrity were assessed by passive permeability, transendothelial electrical resistance (TEER) measurements,and direct visualization of hypoxic cells. The presented protocol offers a distinct advantage inmimicking the intercellular environment of the BBB in vivo, serving as a more realistic in vitro BBB model for developing new therapeutic strategies in the setting of ischemic brain injury.

A Triple Primary Cell Culture Model of the Human Blood-Brain Barrier for Studying Ischemic Stroke In Vitro

Here, we describe the method for establishing a triple cell culture model of the blood-brain barrier based on primary human brain microvascular endothelial cells, astrocytes, and pericytes. This multicellular model is suitable for studies of neurovascular unit dysfunction during ischemic stroke in vitro or for the screening of drug candidates.

用于体外研究缺血性中风的人血脑屏障的三重原代细胞培养模型

Ischemic stroke is a major cause of death and disability worldwide with limited therapeutic options. The neuropathology of ischemic stroke is ...

生物化学

Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week protocol for repetitive hypoxic preconditioning (RHP) induces long-term protection against central nervous system (CNS) injury in a mouse model of focal ischemic stroke. RHP consists of 9 stochastic exposures to hypoxia that vary in both duration (2 or 4 hr) and intensity (8% and 11% O2). RHP reduces infarct volumes, blood-brain barrier (BBB) disruption, and the post-stroke inflammatory response for weeks following the last exposure to hypoxia, suggesting a long-term induction of an endogenous CNS-protective phenotype. The methodology for the dual quantification of infarct volume and BBB disruption is effective in assessing neurovascular protection in mice with RHP or other putative neuroprotectants. Adult male Swiss Webster mice were preconditioned by RHP or duration-equivalent exposures to 21% O2 (i.e. room air). A 60 min transient middle cerebral artery occlusion (tMCAo) was induced 2 weeks following the last hypoxic exposure. Both the occlusion and reperfusion were confirmed by transcranial laser Doppler flowmetry. Twenty-two hr after reperfusion, Evans Blue (EB) was intravenously administered through a tail vein injection. 2 hr later, animals were sacrificed by isoflurane overdose and brain sections were stained with 2,3,5- triphenyltetrazolium chloride (TTC). Infarcts volumes were then quantified. Next, EB was extracted from the tissue over 48 hr to determine BBB disruption after tMCAo. In summary, RHP is a simple protocol that can be replicated, with minimal cost, to induce long-term endogenous neurovascular protection from stroke injury in mice, with the translational potential for other CNS-based and systemic pro-inflammatory disease states.

This protocol describes repetitive hypoxic preconditioning, or brief exposures to systemic hypoxia that reduce infarct volumes and blood-brain barrier disruption following transient middle cerebral artery occlusion in mice. It also details dual quantification of infarct volume and blood-brain barrier disruption after stroke to assess the efficacy of neurovascular protection.

神经血管保护定量继在小鼠重复缺氧预处理和瞬态大脑中动脉闭塞

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week ...

Monitoring Astrocyte Reactivity and Proliferation in Vitro Under Ischemic-Like Conditions

Ischemic stroke is a complex brain injury caused by a thrombus or embolus obstructing blood flow to parts of the brain. This leads to deprivation of oxygen and glucose, which causes energy failure and neuronal death. After an ischemic stroke insult, astrocytes become reactive and proliferate around the injury site as it develops. Under this scenario, it is difficult to study the specific contribution of astrocytes to the brain region exposed to ischemia. Therefore, this article introduces a methodology to study primary astrocyte reactivity and proliferation under an in vitro model of an ischemia-like environment, called oxygen glucose deprivation (OGD). Astrocytes were isolated from 1-4 day-old neonatal rats and the number of non-specific astrocytic cells was assessed using astrocyte selective marker Glial Fibrillary Acidic Protein (GFAP) and nuclear staining. The period in which astrocytes are subjected to the OGD condition can be customized, as well as the percentage of oxygen they are exposed to. This flexibility allows scientists to characterize the duration of the ischemic-like condition in different groups of cells in vitro. This article discusses the timeframes of OGD that induce astrocyte reactivity, hypertrophic morphology, and proliferation as measured by immunofluorescence using Proliferating Cell Nuclear Antigen (PCNA). Besides proliferation, astrocytes undergo energy and oxidative stress, and respond to OGD by releasing soluble factors into the cell medium. This medium can be collected and used to analyze the effects of molecules released by astrocytes in primary neuronal cultures without cell-to-cell interaction. In summary, this primary cell culture model can be efficiently used to understand the role of isolated astrocytes upon injury.

Monitoring Astrocyte Reactivity and Proliferation in Vitro Under Ischemic-Like Conditions

Ischemic stroke is a complex event in which the specific contribution of astrocytes to the affected brain region exposed to oxygen glucose deprivation (OGD) is difficult to study. This article introduces a methodology to obtain isolated astrocytes and study their reactivity and proliferation under OGD conditions.

在缺血样条件下监测星形胶质细胞的反应性和增殖体外

Ischemic stroke is a complex brain injury caused by a thrombus or embolus obstructing blood flow to parts of the brain. This leads to deprivation of ...

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier (BBB). Rats with induced ischemic stroke were established and used as in vivo models to investigate the functional integrity of the BBB. Spectrophotometric detection of Evans blue (EB) in the brain samples with ischemic injury could provide reliable justification for the research and development of novel therapeutic modalities. This method generates reproducible results, and is applicable in any laboratory without a need for special equipment. Here, we present a visualized and technical guideline on the detection of the extravasation of EB following induction of ischemic stroke in rats.

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

The overall goal of this procedure is to provide a highly reproducible technique for in vivo assessment of the blood-brain barrier disruption in rat models of ischemic stroke.

一种体内评价大鼠缺血性中风模型中血脑屏障的破坏

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier ...

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) ...

A Cell Culture Model for Studying the Role of Neuron-Glia Interactions in Ischemia

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent neuronal loss. Numerous evidence suggests that the interaction between glial and neuronal cells exert beneficial effects after an ischemic event. Therefore, to explore potential protective mechanisms, it is important to develop models that allow studying neuron-glia interactions in an ischemic environment. Herein we present a simple approach to isolate astrocytes and neurons from the rat embryonic cortex, and that by using specific culture media, allows the establishment of neuron- or astrocyte-enriched cultures or neuron-glia cultures with high yield and reproducibility.
To study the crosstalk between astrocytes and neurons, we propose an approach based on a co-culture system in which neurons cultured in coverslips are maintained in contact with a monolayer of astrocytes plated in multiwell plates. The two cultures are maintained apart by small paraffin spheres. This approach allows the independent manipulation and the application of specific treatments to each cell type, which represents an advantage in many studies.
To simulate what occurs during an ischemic stroke, the cultures are subjected to an oxygen and glucose deprivation protocol. This protocol represents a useful tool to study the role of neuron-glia interactions in ischemic stroke.

Here, we present a simple approach using specific culture media that allows the establishment of neuron- and astrocyte-enriched cultures, or neuron-glia cultures from the embryonic cortex, with high yield and reproducibility.

研究神经-胶质相互作用在缺血中的作用的细胞培养模型

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent ...

<meta charset="utf8"></head><body>基于荧光染料的小鼠血脑屏障分解可视化

Evaluation of Blood-Brain Barrier Breakdown in a Mouse Model of Mild Traumatic Brain Injury

Fluorescent dyes are used to determine the extent of dye extravasation that occurs due to blood-brain barrier (BBB) breakdown. Labeling with these dyes is a complex process influenced by several factors, such as the concentration of dyes in the blood, permeability of brain vessels, duration of dye extravasation, and reduction in dye concentration in the tissue due to degradation and diffusion. In a mild traumatic brain injury model, exposure to blast-induced shock waves (BSWs) triggers BBB breakdown within a limited time window. To determine the precise sequence of BBB breakdown, Evans blue, and fluorescein isothiocyanate-dextran were injected intravascularly and intracardially into mice at various time points relative to BSW exposure. The distribution of dye fluorescence in brain slices was then recorded. Differences in the distribution and intensity between the two dyes revealed the spatiotemporal sequence of BBB breakdown. Immunostaining of the brain slices showed that astrocytic and microglial responses correlated with the sites of BBB breakdown. This protocol has broad potential for application in studies involving different BBB breakdown models.

<meta charset="utf8"></head><body>轻度创伤性脑损伤小鼠模型中血脑屏障破坏的评价

A method was developed to visualize dye extravasation due to blood-brain barrier (BBB) breakdown by administering two fluorescent dyes to mice at different time points. The use of glycerol as a cryoprotectant facilitated immunohistochemistry on the same sample.

轻度创伤性脑损伤小鼠模型中血脑屏障破坏的评价

Fluorescent dyes are used to determine the extent of dye extravasation that occurs due to blood-brain barrier (BBB) breakdown. Labeling with these dyes is ...

缺氧缺糖再给氧作为

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