Name: an in vivo assessment of blood-brain barrier disruption in a rat model of ischemic stroke
Uploaded: 2018-03-11T14:00:00.000+00:00
Duration: 12 min 19 s
Description: Watch this Scientific Journal Video about An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke at JoVE.com

作者感谢阿尔达比勒医学大学 (阿尔达比勒, 伊朗) 研究的副校长为财政支持 (授予 No: 9607)。

所有的程序都按照阿尔达比勒大学医学研究委员会的指导方针进行动物研究 (道德 ID 号: IR。ARUMS。1394.08). 在这一可视化研究中, 我们使用了由牧场研究所 (伊朗德黑兰) 获得的成年雄性大大鼠 (300-350g)。
1. 麻醉和血流仪
<ol><li>麻醉使用4% 异氟醚, 并保持它与异氟醚 (1-1. 5%) 在一氧化氮 (70% 伏/五) 和氧 (30% v/v) 的混合物在手术期间。</li>
	<li>放置一个麻醉动物在一个反馈控制的加热毯的俯卧姿, 并保持体温在37±0.5 °c 通过一个直肠探头连接到这个加热单元。</li>
	<li>在两只眼睛上涂抹少量的眼膏, 以防止干燥。</li>
	<li>用电动快剪剃去头骨左侧的手术区。使用 betadine 溶液与纱布垫消毒皮肤。用含有70% 乙醇的无菌垫冲洗这个区域, 并重复这两个步骤三循环17。</li>
	<li>为镇痛, 注射0.2 毫升0.5% 布比卡因皮下入手术区域17。</li>
	<li>在头骨上做一个1厘米长的皮肤切口 (从左眼的侧眼角延伸到左耳的底部), 解剖左颞的肌肉以暴露头骨。然后, 创建一个小毛刺孔5毫米侧向和1毫米后向 bregma18 , 以缓解多普勒流量计铅笔探针的尖端位置。</li>
	<li>将大鼠从俯卧位置转到仰卧位, 然后将激光笔探头插入先前钻孔的颅骨盲孔, 监测区域脑血流 (局部脑血)。</li>
	<li>记录每个动物的基线局部脑血, 并将其视为100%。值得注意的是, 每当检测到80% 局部脑血的减少时, 大脑中动脉闭塞 (MCAO) 就开始了19, 20.</li>
</ol>2. 诱导灶性脑缺血
<ol><li>用 betadine 溶液和70% 乙醇对颈部进行剃须, 并对皮肤进行消毒。在手术部位注射0.2 毫升0.5% 布比卡因镇痛17。</li>
	<li>在颈部的腹面上做一个2厘米长的手术切口, 以进入左颈总动脉 (LCCA)。从相邻筋膜和迷走神经中分离 LCCA, 以获得颈外动脉 (ECA) 和颈内动脉的分岔。</li>
	<li>结扎永久 LCCA 或非洲经委会使用5-0 丝缝合和解剖 ICA 自由的水平的翼腭动脉。</li>
	<li>松散地放置在 LCCA 周围的5-0 丝缝合领带, 然后临时 ICA 钳与血管微夹。</li>
	<li>在先前放置松散的领带之前, 在 LCCA 上做一个小切口, 然后在 ICA 的腔内插入4-0 的硅涂层尼龙缝线, 并收紧 LCCA 周围的缝合, 以确保尼龙缝合, 防止血液泄漏。</li>
	<li>从 ICA 中取出血管微夹。然后, 提前一个硅涂层腔内灯丝, 直到观察到明显下降的局部脑血, 表明闭塞的 MCA 起源。在缺血性期结束时 (90 分钟), 通过取出腔内缝合21开始再灌注。</li>
</ol>3. 颈静脉插管和埃文斯蓝 (EB) 注射液
<ol><li>在颈部向中线的左侧做一个1厘米的纵切口, 然后直截了当地解剖掉浅筋膜, 以进入左颈静脉的外侧分支 (轻型货车)。然后, 用5-0 丝缝线永久结扎轻型货车的颅骨末端。在静脉周围松散地放置两条领带, 然后用血管微钳暂时夹住轻型货车的心脏末端。</li>
	<li>在两条缝线之间, 在轻型货车上做一个小切口, 然后将血气血清填充导管插入轻型货车的腔内, 并将其推进约10毫米. 随后, 拧紧静脉周围的连字, 以确保导管的安全并防止出血。注射少量的血清以避免血管塌陷。</li>
	<li>在再灌注期开始时, 在5分钟内缓慢注射 eb 染料 (1 毫升/千克 2% eb 溶液生理盐水)。随后, 通过注射0.5 毫升生理盐水冲洗套管, 从静脉12,22取出注射套管。</li>
	<li>最后, 缝合颈部和头部切口, 注射0.05 毫克/千克丁丙诺啡腹腔 (IP) 和重复注射每6-8 小时在术后镇痛17。</li>
	<li>注入5毫升预热生理盐水 (IP), 以提供恢复笼17中的水合。</li>
	<li>将动物放在装有温度和空调控制的特殊恢复笼中, 并监测动物从麻醉中恢复的情况。</li>
</ol>4. 血脑屏障通透性评估
<ol><li>再灌注24小时后, 用硫喷妥钠深麻醉动物。然后, 通过在胸骨下做一个小洞来打开胸腔。</li>
	<li>在右心房开一个小开口, 在 110 mmHg 的压力下, 通过左心室注射250毫升预热的0.9% 生理盐水 (37 ˚C), 将 EB 从循环中冲洗出去, 直到从心房的正常生理盐水出口变为无色的15。</li>
	<li>立即将大脑从头骨中取出, 并将其置于脑基质中。解剖嗅球和小脑, 然后使用干净的剃刀刀片, 从左 (损伤) 沿中线分开大脑的右半球。</li>
	<li>在2.5 毫升磷酸盐缓冲盐水中分别称量每个半球并融汇它们, 并将其与2.5 毫升的乙酸酸 (60%) 混合, 使用涡旋机进行2分钟。</li>
	<li>离心机的大脑样本为30分钟, 在 1322 x g, 并允许样品冷却在4°c 冰箱10分钟。</li>
	<li>使用上清液估计在 610nm23 的分光光度计的 EB 吸光度。</li>
	<li>根据标准曲线计算 EB 浓度, 并将结果表达为脑组织的µg/g。</li>
</ol>5. 电子束标准曲线的制作
<ol><li>在5毫升磷酸盐缓冲盐水中制备10个 eb 的样品溶液, 其浓度为 eb 的1-10 µg。</li>
	<li>使用分光光度计测量每样样品的吸光度值 610 nm。</li>
	<li>使用电子表格制作散点图, 并在 X 轴上显示 EB 浓度和 Y 轴上的吸光度值 (图 1)。</li>
	<li>用方程定义曲线的线性趋势线。然后, 用它来获得样品的 EB 浓度。</li>
	<li>报告最终结果的 EB 渗出作为µg/g 的脑组织重量。</li>
</ol>6. 假操作
<ol><li>对假手术组的大鼠进行相同的手术处理 (包括在颈部和 EB 注射液中进行切口), 但排除 MCAO。</li>
</ol>

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K., Kleinschnitz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+ischemic+stroke+and+their+application+in+clinical+research.">Animal models of ischemic stroke and their application in clinical research.</a> Drug Des Devel Ther. 9, 3445-3454 (2015).</li><li>Noor, R., Wang, C. X., Shuaib, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+hyperthermia+on+infarct+volume+in+focal+embolic+model+of+cerebral+ischemia+in+rats.">Effects of hyperthermia on infarct volume in focal embolic model of cerebral ischemia in rats.</a> Neurosci Lett. 349 (2), 130-132 (2003).</li><li>Shin, H. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mild+induced+hypertension+improves+blood+flow+and+oxygen+metabolism+in+transient+focal+cerebral+ischemia.">Mild induced hypertension improves blood flow and oxygen metabolism in transient focal cerebral ischemia.</a> Stroke. 39 (5), 1548-1555 (2008).</li><li>Bottiger, B. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+cerebral+ischemia+due+to+cardiocirculatory+arrest+in+mice+causes+neuronal+degeneration+and+early+induction+of+transcription+factor+genes+in+the+hippocampus.">Global cerebral ischemia due to cardiocirculatory arrest in mice causes neuronal degeneration and early induction of transcription factor genes in the hippocampus.</a> Brain Res Mol Brain Res. 65 (2), 135-142 (1999).</li></ol>

作者没有什么可透露的。

迄今为止, 各种方法, 如显影和检测放射性示踪剂24,25, 免疫荧光显微镜26,27, EB 渗出技术20, 23已被用来评估血脑屏障损伤。EB 染料强烈能够绑定到血清白蛋白, 并作为追踪器, 以检测血管渗漏和量化的 BBB 击穿11,28,<su...

血管源性脑水肿由于血脑屏障 (BBB) 中断仍然是缺血性中风的重要并发症, 也是中风患者存活率的主要决定因素1, 2.血脑屏障 (BBB) 由脑毛细血管内皮细胞 (BCECs) 形成, 由不同的神经血管成分组成 (例如, BCECs、毛细血管、星形胶质和神经细胞之间的紧密连接3), 提供了一种中枢神经系统 (CNS) 与外周血循环之间的专门和动态接口4,5。诸如缺血再灌注损伤等侮辱可能扰乱脑屏障功能完整性, 导致循环白细胞随后渗入大脑实质, 最终引发脑部炎症和原发性脑损伤。6,7. 需要动物模型来精确检测中风发生后的脑屏障功能障碍。这些模型对于研究潜在的病理生理学机制和引入新的神经保护策略具有重要意义。体外基于细胞培养的脑屏障模型已得到高度发展, 并用于生理病理的分子研究(bbb) 8, 9, 10.然而, 在这方面,在体内动物模型, 它产生的脑屏障的缺血性损伤类似于人类的临床条件, 也是非常值得的。定量检测埃文斯蓝 (EB) 的渗出是一种公认和敏感的技术, 已用于评估脑屏障完整性和功能的神经退行性疾病, 包括缺血性中风11,12,13,14. 该方法成本效益高, 可行, 重现性好, 完全适用于任何实验实验室。它的实现不需要先进的设备, 如放射性示踪剂15或磁共振成像 (MRI)16, 这是其他方法的先决条件。本文对缺血性脑卒中大鼠模型中 EB 渗出进行了脑屏障的基本技术过程的综合演示。

<table><tbody><tr><td>Isoflurane</td><td>Piramal</td><td>AWN 34041100</td><td>20 - 25 &amp;deg;C</td></tr><tr><td>2,3,5-Triphenyltetrazolium chloride (TTC)</td><td>Molekula</td><td>31216368</td><td>4 years</td></tr><tr><td>Sprague&amp;ndash;Dawley rats&amp;nbsp;</td><td>Pasture Institute (Tehran, Iran)</td><td></td><td>300-350g</td></tr><tr><td>Evans Blue&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>314-13-6</td><td></td></tr><tr><td>Trichloroacetic acid&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>76-03-9</td><td>2 years</td></tr><tr><td>Bupivacaine HCl (0.5%)</td><td>Delpharm Tours</td><td></td><td>below&amp;nbsp; 25 &amp;deg;C</td></tr><tr><td>Bupernorphine</td><td>Exir (Iran)</td><td></td><td></td></tr><tr><td>Sodium Carbonate</td><td>Sigma-Aldrich&amp;nbsp;</td><td>497-19-8</td><td></td></tr><tr><td>Sodium chloride&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>7647-14-5</td><td></td></tr><tr><td>Di- Sodium hydrogen phosphate</td><td>EMD Millipore&amp;nbsp;</td><td>231-448-7</td><td></td></tr><tr><td>Potassium chloride</td><td>Sigma-Aldrich&amp;nbsp;</td><td>&amp;nbsp;7447-40-7</td><td></td></tr><tr><td>Ethanol&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>64-17-5</td><td></td></tr><tr><td>silicone(Xantopren)</td><td>Heraeus</td><td>EN ISO 4823</td><td></td></tr><tr><td>Activator universal plus</td><td>Heraeus</td><td>66037445</td><td></td></tr><tr><td>Micro-Dissecting forceps</td><td>Stoelting</td><td>52100-41</td><td></td></tr><tr><td>Spring Scisors</td><td>Stoelting</td><td>52130-00</td><td></td></tr><tr><td>Operating&amp;nbsp; Scissors</td><td>Roboz</td><td>52140-70</td><td></td></tr><tr><td>Brain matrix&amp;nbsp;</td><td>Stoelting</td><td>51390</td><td></td></tr><tr><td>Anesthesia Machine for Small Animals |</td><td>&amp;nbsp;Kent Scientific</td><td>SS-01</td><td></td></tr><tr><td>Power Lab system</td><td>AD Instruments</td><td>ML880</td><td></td></tr><tr><td>Laser Doppler flowmeter</td><td>AD Instruments</td><td>ML191</td><td></td></tr><tr><td>Heating feed back system</td><td>Harvard Appratus</td><td>72-7560</td><td></td></tr><tr><td>Vascular micro clamp</td><td>FineScience Tools</td><td>18055-03</td><td></td></tr><tr><td>Silk 5-0 suture thread</td><td>Ethicon</td><td>682G</td><td></td></tr><tr><td>Ethilon 4-0 suture thread&amp;nbsp;</td><td>Ethicon</td><td>EH6740G</td><td></td></tr></tbody></table>

an in vivo assessment of blood-brain barrier disruption in a rat model of ischemic stroke

缺血性中风导致血管源性脑水肿和随后的原发性脑损伤, 这是通过破坏血脑屏障 (BBB) 介导的。建立了诱导缺血性中风大鼠, 并作为体内模型对脑屏障功能完整性进行了研究。分光光度法检测脑标本中的埃文斯蓝 (EB), 可以为新的治疗方式的研究和开发提供可靠的依据。这种方法产生可重现的结果, 并适用于任何实验室, 不需要特殊设备。在此, 我们提出了一个可视化的技术指南, 以检测的 EB 渗出后, 诱导的缺血性中风的大鼠。

右半球 EB 水平与假手术大鼠左半球无显著性差异 (分别为 1.06 0.1 µg/克和 1.1 @ 0.09 µg/克)。如图 2 a-2 b所示, 在缺血性大鼠的左半球, 诱导短暂缺血 (90 分钟缺血/24 小时再灌注) 导致 EB 水平显著差异 (10.41 @ 0.84 µg/g, p < 0.001), 与各自半球在假操作的老鼠。总的来说, 这些发现表明, 在正常情况下, eb 不能轻易地越过脑屏障进入大脑实质和脑缺血性侮辱诱发 EB ?...

Watch this Scientific Journal Video about An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke at JoVE.com

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

本程序的总体目标是为在体内评估缺血性中风模型大鼠血脑屏障中断提供一种高度可重复的技术。

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

an-vivo-assessment-blood-brain-barrier-disruption-rat-model-ischemic

Research

JoVE Journal

Neuroscience

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

11.3K 次观看.  Ardabil University of Medical Sciences. 本程序的总体目标是为在体内评估缺血性中风模型大鼠血脑屏障中断提供一种高度可重复的技术。

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

Focal Cerebral Ischemia Model by Endovascular Suture Occlusion of the Middle Cerebral Artery in the Rat

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable clinical state; in the development of animal models of focal ischemia, however, achieving reproducibility of experimentally induced infarct volume is essential. The rat is a widely used animal model for stroke due to its relatively low animal husbandry costs and to the similarity of its cranial circulation to that of humans2,3. In humans, the middle cerebral artery (MCA) is most commonly affected in stroke syndromes and multiple methods of MCA occlusion (MCAO) have been described to mimic this clinical syndrome in animal models. Because recanalization commonly occurs following an acute stroke in the human, reperfusion after a period of occlusion has been included in many of these models. In this video, we demonstrate the transient endovascular suture MCAO model in the spontaneously hypertensive rat (SHR). A filament with a silicon tip coating is placed intraluminally at the MCA origin for 60 minutes, followed by reperfusion. Note that the optimal occlusion period may vary in other rat strains, such as Wistar or Sprague-Dawley. Several behavioral indicators of stroke in the rat are shown. Focal ischemia is confirmed using T2-weighted magnetic resonance images and by staining brain sections with 2,3,5-triphenyltetrazolium chloride (TTC) 24 hours after MCAO.

Surgical induction of ischemic brain damage in the rat is a widely used model for stroke research. Here we demonstrate the induction of focal cerebral ischemia by occlusion of the middle cerebral artery. Visualization of the resulting infarct by histological staining and magnetic resonance imaging is also shown.

焦距脑缺血模型大鼠大脑中动脉的血管内缝合闭塞

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable ...

科研

神经科学

Rat Model of Blood-brain Barrier Disruption to Allow Targeted Neurovascular Therapeutics

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain barrier1. The barrier selectively excludes molecules from crossing between the blood and the brain based upon their size and charge. This function can impede the delivery of therapeutics for neurological disorders. A number of chemotherapeutic drugs, for example, will not effectively cross the blood-brain barrier to reach tumor cells2. Thus, improving the delivery of drugs across the blood-brain barrier is an area of interest.
The most prevalent methods for enhancing the delivery of drugs to the brain are direct cerebral infusion and blood-brain barrier disruption3. Direct intracerebral infusion guarantees that therapies reach the brain; however, this method has a limited ability to disperse the drug4. Blood-brain barrier disruption (BBBD) allows drugs to flow directly from the circulatory system into the brain and thus more effectively reach dispersed tumor cells. Three methods of barrier disruption include osmotic barrier disruption, pharmacological barrier disruption, and focused ultrasound with microbubbles. Osmotic disruption, pioneered by Neuwelt, uses a hypertonic solution of 25% mannitol that dehydrates the cells of the blood-brain barrier causing them to shrink and disrupt their tight junctions. Barrier disruption can also be accomplished pharmacologically with vasoactive compounds such as histamine5 and bradykinin6. This method, however, is selective primarily for the brain-tumor barrier7. Additionally, RMP-7, an analog of the peptide bradykinin, was found to be inferior when compared head-to-head with osmotic BBBD with 25% mannitol8. Another method, focused ultrasound (FUS) in conjunction with microbubble ultrasound contrast agents, has also been shown to reversibly open the blood-brain barrier9. In comparison to FUS, though, 25% mannitol has a longer history of safety in human patients that makes it a proven tool for translational research10-12.
In order to accomplish BBBD, mannitol must be delivered at a high rate directly into the brain's arterial circulation. In humans, an endovascular catheter is guided to the brain where rapid, direct flow can be accomplished. This protocol models human BBBD as closely as possible. Following a cut-down to the bifurcation of the common carotid artery, a catheter is inserted retrograde into the ECA and used to deliver mannitol directly into the internal carotid artery (ICA) circulation. Propofol and N2O anesthesia are used for their ability to maximize the effectiveness of barrier disruption13. If executed properly, this procedure has the ability to safely, effectively, and reversibly open the blood-brain barrier and improve the delivery of drugs that do not ordinarily reach the brain 8,13,14.

Blood-brain barrier disruption aids the delivery of certain drugs to the brain. Mannitol delivered intra-arterially shrinks cells surrounding blood vessels in order to physically disrupt the barrier.

允许有针对性的大鼠模型血 - 脑屏障破坏神经血管治疗

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain ...

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 ...

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 ...

Assessment of Blood-brain Barrier Permeability by Intravenous Infusion of FITC-labeled Albumin in a Mouse Model of Neurodegenerative Disease

Disruption of blood-brain barrier (BBB) integrity is a common feature for different neurological and neurodegenerative diseases. Although the interplay between perturbed BBB homeostasis and the pathogenesis of brain disorders needs further investigation, the development and validation of a reliable procedure to accurately detect BBB alterations may be crucial and represent a useful tool for potentially predicting disease progression and developing targeted therapeutic strategies.
Here, we present an easy and efficient procedure for evaluating BBB leakage in a neurodegenerative condition like that occurring in a preclinical mouse model of Huntington disease, in which defects in the permeability of BBB are clearly detectable precociously in the disease. Specifically, the high molecular weight fluorescein isothiocyanate labelled (FITC)-albumin, which is able to cross the BBB only when the latter is impaired, is acutely infused into a mouse jugular vein and its distribution in the vascular or parenchymal districts is then determined by fluorescence microscopy.
Accumulation of green fluorescent-albumin in the brain parenchyma functions as an index of aberrant BBB permeability and, when quantitated by using Image J processing software, is reported as Green Fluorescence Intensity.

In this study, we present an easy and efficient procedure for evaluating the disruption of the blood-brain barrier under neurodegenerative conditions. To achieve our objective, we infused high molecular weight fluorescein isothiocyanate labelled (FITC)-albumin into mouse jugular vein and evaluated its leakage into the brain parenchyma by fluorescence microscopy.

FITC 标记白蛋白静脉滴注对神经退行性疾病模型血脑屏障通透性的评价

Disruption of blood-brain barrier (BBB) integrity is a common feature for different neurological and neurodegenerative diseases. Although the interplay ...

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains brain homeostasis. The principal sites of the barrier are endothelial cells of the brain capillaries whose barrier function results from tight intercellular junctions and efflux transporters expressed on the plasma membrane. This function is regulated by pericytes and astrocytes that together form the neurovascular unit (NVU). Several neurological diseases such as stroke, Alzheimer&#39;s disease (AD), brain tumors are associated with an impaired BBB function. Assessment of the BBB permeability is therefore crucial in evaluating the severity of the neurological disease and the success of the treatment strategies employed.
We present here a simple yet robust permeability assay that have been successfully applied to several mouse models both, genetic and experimental. The method is highly quantitative and objective in comparison to the tracer fluorescence analysis by microscopy that is commonly applied. In this method, mice are injected intraperitoneally with a mix of aqueous inert fluorescent tracers followed by anesthetizing the mice. Cardiac perfusion of the animals is performed prior to harvesting brain, kidneys or other organs. Organs are homogenized and centrifuged followed by fluorescence measurement from the supernatant. Blood drawn from the cardiac puncture just before perfusion serves for normalization purpose to the vascular compartment. The tissue fluorescence is normalized to the wet weight and serum fluorescence to obtain a quantitative tracer permeability index. For additional confirmation, the contralateral hemi-brain preserved for immunohistochemistry can be utilized for tracer fluorescence visualization purposes.

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Here we present a mouse brain vascular permeability assay using intraperitoneal injection of fluorescent tracers followed by perfusion that is applicable to animal models of blood-brain barrier dysfunction. One hemi-brain is used for assessing permeability quantitatively and the other for tracer visualization/immunostaining. The procedure takes 5 - 6 h for 10 mice.

用荧光标记示踪剂对小鼠血脑屏障通透性的体内检测

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains ...

Measuring Post-Stroke Cerebral Edema, Infarct Zone and Blood-Brain Barrier Breakdown in a Single Set of Rodent Brain Samples

One of the most common causes of morbidity and mortality worldwide is ischemic stroke. Historically, an animal model used to stimulate ischemic stroke involves middle cerebral artery occlusion (MCAO). Infarct zone, brain edema and blood-brain barrier (BBB) breakdown are measured as parameters that reflect the extent of brain injury after MCAO. A significant limitation to this method is that these measurements are normally obtained in different rat brain samples, leading to ethical and financial burdens due to the large number of rats that need to be euthanized for an appropriate sample size. Here we present a method to accurately assess brain injury following MCAO by measuring infarct zone, brain edema and BBB permeability in the same set of rat brains. This novel technique provides a more efficient way to evaluate the pathophysiology of stroke.

This protocol describes a novel technique of measuring the three most important parameters of ischemic brain injury on the same set of rodent brain samples. Using only one brain sample is highly advantageous in terms of ethical and economic costs.

测量一组啮齿动物脑样本中的中风后脑水肿、梗塞区和血脑屏障破裂

One of the most common causes of morbidity and mortality worldwide is ischemic stroke. Historically, an animal model used to stimulate ischemic stroke ...

Real-Time Monitoring and Modulation of Blood Pressure in a Rabbit Model of Ischemic Stroke

Control of blood pressure, in terms of both absolute values and its variability, affects outcomes in ischemic stroke patients. However, it remains challenging to identify the mechanisms that lead to poor outcomes or evaluate measures by which these effects can be mitigated because of the prohibitive limitations inherent to human data. In such cases, animal models can be utilized to conduct rigorous and reproducible evaluations of diseases. Here we report refinement of a previously described model of ischemic stroke in rabbits that is augmented with continuous blood pressure recording to assess the impacts of modulation on blood pressure. Under general anesthesia, femoral arteries are exposed through surgical cutdowns to place arterial sheaths bilaterally. Under fluoroscopic visualization and roadmap guidance, a microcatheter is advanced into an artery of the posterior circulation of the brain. An angiogram is performed by injecting the contralateral vertebral artery to confirm occlusion of the target artery. With the occlusive catheter remaining in position for a fixed duration, blood pressure is continuously recorded to allow for tight titration of blood pressure manipulations, whether through mechanical or pharmacological means. At the completion of the occlusion interval, the microcatheter is removed, and the animal is maintained under general anesthesia for a prescribed length of reperfusion. For acute studies, the animal is then euthanized and decapitated. The brain is harvested and processed to measure the infarct volume under light microscopy and further assessed with various histopathological stains or spatial transcriptomic analysis. This protocol provides a reproducible model that can be utilized for more thorough preclinical studies on the effects of blood pressure parameters during ischemic stroke. It also facilitates effective preclinical evaluation of novel neuroprotective interventions that might improve care for ischemic stroke patients.

Continuous arterial blood pressure recording allows the investigation of impacts of various hemodynamic parameters. This report demonstrates the application of continuous arterial blood pressure monitoring in a large animal model of ischemic stroke for determination of stroke pathophysiology, impact of different hemodynamic factors, and the assessment of novel treatment approaches.

缺血性脑卒中兔模型中血压的实时监测和调节

Control of blood pressure, in terms of both absolute values and its variability, affects outcomes in ischemic stroke patients. However, it remains ...

<meta charset="utf8"></head><body>免疫荧光染色研究大鼠 MCAO 卒中模型中的细胞死亡

Evaluating Cell Death Signaling by Immunofluorescence in a Rat Model of Ischemic Stroke

Stroke is a leading cause of death and disability worldwide. Most cases of stroke are ischemic and result from the occlusion of the middle cerebral artery (MCA). Current pharmacological approaches for the treatment of ischemic stroke are limited; therefore, novel therapies providing effective neuroprotection against ischemic injury following stroke are urgently needed. In the brain tissue following ischemic stroke, the area of the ischemic penumbra is salvageable but at risk of progressing to irreversible damage. The penumbral area surrounding the infarcted core is a conceptual target for neuroprotection. Since the blood flow is partially maintained in the penumbral area, neuronal and non-neuronal cells in this area transiently survive after stroke, and these cells that are still viable may be rescued by appropriate medical interventions. Understanding the pathophysiology of the penumbra is important in the development of neuroprotective therapies because the cell death pathways activated in the ischemic penumbra may indicate therapeutic targets, such as RUNX1 and cathepsins. These protein targets functioning as mediators of programmed cell death can be further exploited in translational research. With moderate size and similarity to the human brain, the in vivo rat model of MCA occlusion (MCAO) mimics the human ischemic stroke and offers an applicable tool to investigate the penumbral pathology, examine the cell death signaling, and evaluate the effects of potential targets in the context of MCAO. Here, we describe how to induce MCAO in rats and how to perform immunofluorescence staining for the detection of cell death signaling in the rat brain following MCAO.

<meta charset="utf8"></head><body>通过免疫荧光评估缺血性卒中大鼠模型中的细胞死亡信号

Rat in vivo models are indispensable tools to investigate the pathological mechanisms and therapeutic targets for ischemic stroke. This work will describe performing immunofluorescence staining in infarcted brain slices following middle cerebral artery occlusion (MCAO) in rats.

通过免疫荧光评估缺血性卒中大鼠模型中的细胞死亡信号

Stroke is a leading cause of death and disability worldwide. Most cases of stroke are ischemic and result from the occlusion of the middle cerebral artery ...