笔者想感谢他们的帮助与照顾动物肖纳赖德奥特格罗斯和亚历山德拉Garces，平吴为她的技术援助。由国立卫生研究院下，没有为这项工作提供了支持。 EB003268，加拿大研究主席的计划。

1。超声和​​MRI设置在这项研究中使用了核磁共振兼容的三轴聚焦超声系统（聚焦超音波仪器公司，多伦多，安大略，加拿大）。使用两种不同的超声换能器：内部构造551.5千赫球为中心的传感器（曲率半径为60毫米，外直径为75毫米，内直径为20毫米），和1.503兆赫，8部门阵列综合作为一个单一的元素球聚焦换能器（FN = 0.8，光圈= 10厘米）驱动压电陶瓷水听器（Imasonic公司，Voray sur-L&#39;Orgnon的，法国）。一个MRI兼容氟碳接收8用来记录声波发射时的551.5千赫传感器使用。如果使用不同的设备，以下建议： <ol><li>选择适当，在中心范围内的0.25兆赫-1.5兆赫频率和F-数小于或等于1（半径曲率/光圈）校准的超声换能器。 </li><li>将水洗澡FIlled与温暖，脱气，去离子水床，一个1.5 T或3个T磁共振。洗澡的水应该有一个顶级板，可容纳动物。超声换能器安装在水箱中的MRI兼容的三轴定位阶段或系统9，面对水面。 </li><li>传感器电缆运行超声波驱动设备通过渗透面板。 </li><li>使用函数发生器和功率放大器产生的换能器驱动信号。使用外部匹配电路，以尽量减少所反映的电力。 </li><li>了解传感器的焦距，在水面上放置传感器的焦点和超声水面产生一个可见的喷泉。步骤1.6和1.7描述了如何注册使用生成的水喷泉的焦点。一个更精确的定位的重点，其中包括超声分散凝胶和成像焦点的温度与MRI海拔热吸收的方法，可以发现在9 &lt;/ SUP&gt;。 </li><li>标记使用标记，这将是可见的磁共振图像上的焦点位置。这可以用一盘中心孔放置在重点时，将水补。充水孔将在MRI可见，将提供两架飞机的焦点坐标，而在坐标轴方向的传感器，可以从水面决定。 </li><li>使用3飞机航向序列，图像的焦点标记和记录的重点在MRI坐标系的位置。 </li><li>为了监测声发射，装载磁共振兼容的的听音8，作为一个被动的空蚀探测器在冲着传感器焦点水浴（PCD）的使用，或使用一个集成的水听器的传感器。 </li><li> PCD的电缆运行范围卡是足够快的捕捉阵阵高达10毫秒PRFs 2赫兹（如ATS460了AlazarTech，潘特克莱尔，魁北克，加拿大）。电缆必须接地的penetration面板或射频屏蔽以减少噪声。 </li></ol> 2。动物的制备<ol><li>麻醉动物isofluorane气体。由于isofluorane BBB级中断10的效果，应采取动物断了气实验开始前10分钟。在麻醉开始，以保​​护角膜干燥或其他人身伤害的地方每眼眼润滑油膏。 </li><li>使用电动剃须刀刮脸毛皮动物的头部和颈部的上方，然后取出剩下的皮毛，使用脱毛霜（例如奈尔，教会和德怀特有限公司，普林斯顿，新泽西州，美国），并用温和的肥皂冲洗头皮和水。 </li><li>准备与优碘皮肤由bridine洗擦洗尾部。执行酒精的最后wipedown在以可视化的尾静脉。 </li><li> 3路阀22压力表导管插入到尾静脉和冲洗用肝素/生理盐水混合液（33 U / ml的），以防止血凝块形成在导管。 </li><li>交付（40-50毫克/公斤氯胺酮，10毫克/公斤甲苯噻嗪）注射麻醉剂，通过肌肉注射和动物从isofluorane删除。 </li><li>将头顶部的联系，通过在顶部板（图）孔水浴超声定位系统对麻醉动物仰卧。少量的超声波凝胶可能应用到动物的头顶部尽量减少被困airbubbles的的机会。 </li><li>腿磁带定位系统。头可在地方举行，使用咬一口吧，如果有的话，或磁带放在整个下巴坚决。 </li><li>涵盖了动物用毛巾和循环水毯，以保持温暖。 </li></ol> 3。目标选择<ol><li>收购基准轴向ţ2加权和T 1加权MR脑图像。如果使用1.5 T的MRI和专用头大小的射频接收表面线圈，适合SC一个参数可能是： T 2加权FSE，则TE = 60毫秒，TR = 2000毫秒 T 1加权：法兰克福，TE = 10毫秒，TR = 500毫秒</li><li>选择目标从T 2加权扫描，避免脑室和大脑中线，选择中脑的深度。 </li><li>将传感器焦点移动到目标位置。 </li></ol> 4。微泡的制备 DEFINITY微气泡（Lantheus医学影像，MA，美国）用于诱导BBBD 2,5,7微泡介导的FUS的几组。其他类型的气泡适当的剂量，可以发现在文献11,12。 <ol><li>激活DEFINITY微泡，并慢慢地绘制成使用18号针头注射器1毫升体积小。 </li><li>取出轻轻来回移动柱塞空气被困注射器。不要挖掘注射器，因为这可以打破的气泡。 </li><li>在正常稀释的DEFINITY<html>一个10:1的盐水比例的生理盐水的注射器缓缓注入所需体积的DEFINITY Definty生理盐水。如果输液的交付使用，DEFINITY可以进一步稀释，以50:1或100:1。 </li><li>轻轻倒置注射器彻底混合DEFINITY和生理盐水，直至达到甚至外观。立即注射前注射器轻轻翻转也可能是必要的，如果泡沫已经开始暂停浮动。 </li><li> 0.02毫升/的DEFINITY公斤，涨幅为0.2毫升/千克的溶液（10:1稀释）为基础计算出所需的剂量体积。 </li></ol> 5。超声交货<ol><li>超声参数设置，使用低占空比阵阵，不连续波超声处理。在0.5兆赫适当的超声参数的原位压力0.23兆帕，10毫秒脉冲重复频率1赫兹，2分钟阵阵。总超声时候。在1.5兆赫适当的原位压力下降约0.45-0.5兆帕。 </li></ol>_content“&gt;适当的压力，在不同的频率可估计使用机械指数0.46 13。 <ol start="2"><li>检查动物的头仍然是耦合的水。 </li><li>注入的微气泡，慢慢进入尾静脉导管，并用0.5毫升生理盐水冲洗。开始超声同时开始注射。 </li></ol> 6。 MRI评价治疗效果<ol><li>继超声，注入磁共振造影剂（如0.2毫升/公斤的OmniScan，GE医疗）经尾静脉导管由0.5毫升生理盐水冲洗。 </li><li> T 1加权成像对比高峰期已过，直到执行。超声已成功地破坏的网站会比周围组织表现出更大的增强。 </li><li>执行T 2加权成像检查水肿。在超声网站的高信号水肿的指标。 </li></ol> 7。代表结果磁共振造影剂，可透过血脑屏障的成功交付使用聚焦超声和循环微泡。图2显示了典型的前和后的FUS T 1 W图象。图2B显示对比度增强（CE）T 1-W四个超声波处理地点不同的焦距开口的形象。超声位置1和2显示特别明亮的增强。在图3位置1和2还可以看出对应的T-W高信号，表明水肿。 2程度的T-W水肿，有时可以更容易地可视化上矢状切片。图4显示了CE-T的1-W和T-W矢状切片通过超声地点1和3。水肿是在位置1，但不是位置3可见。 捕获的声发射数据（图5）频谱分析可能会显示谐波发射和/或子/超稳定蚀发生时的谐波发射。哈蒙ICS也可以组织产生的非线性，而子和高次谐波的排放只能出现泡沫活动14。在更高的压力，也可以检测到表明惯性蚀宽带排放。然而，这些已与红细胞外渗和微损伤较大数额比没有惯性空化的超声处理11。 使用超声波频率更高更本地化的开口，由于较小的焦斑大小的结果。图6显示了更高的频率，可用于创建开放的较小的区域。这使得用较少的近头骨影响大脑中的作用的调查。 <img alt="图1。" src="/files/ftp_upload/3555/3555fig1.jpg" /> 图1实验装置。 <sp一个类=“的pdflinebreak”&gt; <img alt="图2。" src="/files/ftp_upload/3555/3555fig2.jpg" /> 图2。前（左）后（右）治疗T 1-W显示在四个超声波位置增强大鼠脑图像。 <img alt="图3。" src="/files/ftp_upload/3555/3555fig3.jpg" /> 图3。前（左）后（右）治疗T 2-W显示ţ2-W超声位置1和2的水肿（图2相同的动物）老鼠大脑的图像。 <img alt="图4。" src="/files/ftp_upload/3555/3555fig4.jpg" /> 图4。后处理矢状ţ1-W（左）和T-W（右）从相同的大鼠脑影像图。 2和3。在位置1开幕（左）及其与T-W水肿（右）。位置3显示了开幕式（左），但没有T-W水肿。 </P&gt; <img alt="图5。" src="/files/ftp_upload/3555/3555fig5.jpg" /> 图5。从捕获的数据在一个单一的10毫秒551.5千赫突发的频率频谱。基本频率（F 0）以及谐波（2 F 0）和子/高次谐波（1.5 0.5 F 0，F 0）是可见的。 <img alt="图6。" src="/files/ftp_upload/3555/3555fig6.jpg" /> 图6。后处理CE-T的1-W轴（左）和矢状（右）从1.503兆赫超声在四个地点在大鼠脑图像。在这个频率BBB开口看到更多的本地化。

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K.许尼宁和普拉河的FUS仪器的联合创始人，公司·乔普拉，A. Waspe和光许尼宁的FUS仪器的股东公司K.许尼宁收到的FUS文书，公司的研究支持

动物和微泡的制备此过程中最关键的环节。必须完全去除头发上的动物的头，以避免衰减的超声波束。血脑屏障可以isofluorane麻醉下打乱，但是，它变得更加难以实现一致的开幕式。 应始终与保健和小计处理的微气泡，大口径的针头必须使用绘图时，为了避免打破他们。同样，可以合理地用在尾静脉最小的压力表导管应采用建议（22号）。如果需要一个较小的导管，以实现妥?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>小动物聚焦超声系统</td><td>公司的FUS仪器， </td><td> RK-100 </td></tr><tr><td> DEFINITY </td><td> lantheus医学成像</td><td></td></tr><tr><td> OmniScan的</td><td> GE医疗集团</td><td></td></tr></tbody></table>

mri-guided disruption of the blood-brain barrier using transcranial focused ultrasound in a rat model

聚焦超声（FUS）破坏的血脑屏障（BBB）的是一个日益调查的技术绕过血脑屏障1-5。血脑屏障脑部疾病的药物治疗是一个重大的障碍，因为它限制从血管进入脑组织分子小于约500大小6大通过分子。引起血脑屏障破坏的FUS（BBBD）是暂时的和可逆的4对化学品的优势意味着，诱导高度本地化BBBD的。的FUS的诱导BBBD提供的治疗药物对大脑，组织足够的浓度，否则不会交付广泛的影响调查的一种手段。而广泛的超声参数已被证明成功的破坏血脑屏障2,5,7，在实验过程中有几个关键的步骤，以确保准确定位成功中断。这protocOL轮廓如何实现诱导的大鼠模型BBBD MRI引导下聚焦超音波，一个关键的动物实验的准备和气泡的处理步骤的重点。

Watch this Scientific Journal Video about MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model at JoVE.com

MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model

微泡介导的重点超声破坏血脑屏障（BBB）的是一个很有前途的技术，针对大脑中的非侵入性给药<sup&gt; 1-3</sup&gt;。该协议概述MRI引导下经颅血脑屏障破坏大鼠模型的实验过程。

MRI引导下的破坏血脑屏障在大鼠模型经颅聚焦超声

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a ...

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17.8K 次观看.  Sunnybrook Research Institute. 微泡介导的重点超声破坏血脑屏障（BBB）的是一个很有前途的技术，针对大脑中的非侵入性给药 1-3。该协议概述MRI引导下经颅血脑屏障破坏大鼠模型的实验过程。

视频: MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

利用磁共振成像，容积电脑断层扫描及超音波的裸鼠乳腺癌骨转移大鼠模型血管生成的多模态成像

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

科研

医学

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 ...

A Model of Chronic Nutrient Infusion in the Rat

Chronic exposure to excessive levels of nutrients is postulated to affect the function of several organs and tissues and to contribute to the development of the many complications associated with obesity and the metabolic syndrome, including type 2 diabetes. To study the mechanisms by which excessive levels of glucose and fatty acids affect the pancreatic beta-cell and the secretion of insulin, we have established a chronic nutrient infusion model in the rat. The procedure consists of catheterizing the right jugular vein and left carotid artery under general anesthesia; allowing a 7-day recuperation period; connecting the catheters to the pumps using a swivel and counterweight system that enables the animal to move freely in the cage; and infusing glucose and/or Intralipid (a soybean oil emulsion which generates a mixture of approximately 80% unsaturated/20% saturated fatty acids when infused with heparin) for 72 hr. This model offers several advantages, including the possibility to finely modulate the target levels of circulating glucose and fatty acids; the option to co-infuse pharmacological compounds; and the relatively short time frame as opposed to dietary models. It can be used to examine the mechanisms of nutrient-induced dysfunction in a variety of organs and to test the effectiveness of drugs in this context.

A protocol for chronic infusions of glucose and Intralipid in rats is described. This model can be used to study the impact of nutrient excess on organ function and physiological parameters.

在大鼠模型慢性营养输液

Chronic exposure to excessive levels of nutrients is postulated to  affect the function of several organs and tissues and to contribute to the  ...

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 ...

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 ...

Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound

We present a protocol for measuring in vivo aortic stiffness in mice using high-resolution ultrasound imaging. Aortic diameter is measured by ultrasound and aortic blood pressure is measured invasively with a solid-state pressure catheter. Blood pressure is raised then lowered incrementally by intravenous infusion of vasoactive drugs phenylephrine and sodium nitroprusside. Aortic diameter is measured for each pressure step to characterize the pressure-diameter relationship of the ascending aorta. Stiffness indices derived from the pressure-diameter relationship can be calculated from the data collected. Calculation of arterial compliance is described in this protocol.
This technique can be used to investigate mechanisms underlying increased aortic stiffness associated with cardiovascular disease and aging. The technique produces a physiologically relevant measure of stiffness compared to ex vivo approaches because physiological influences on aortic stiffness are incorporated in the measurement. The primary limitation of this technique is the measurement error introduced from the movement of the aorta during the cardiac cycle. This motion can be compensated by adjusting the location of the probe with the aortic movement as well as making multiple measurements of the aortic pressure-diameter relationship and expanding the experimental group size.

Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound

We describe a technique for measuring aortic stiffness from its pressure-diameter relationship in vivo in mice. Aortic diameter is recorded by ultrasound and aortic pressure is measured invasively with a solid-state pressure catheter. Blood pressure is changed incrementally and the resulting diameter is measured.

测量升主动脉刚度<em&gt;在体内</em&gt;小鼠使用超声

We present a protocol for measuring in vivo aortic stiffness in mice using high-resolution ultrasound imaging. Aortic diameter is measured by ultrasound ...

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

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic cerebrovascular accidents account for 80% of all strokes. A common cause of IR injury is the rapid inflow of fluids following an acute/chronic occlusion of blood, nutrients, oxygen to the tissue triggering the formation of free radicals.
Ischemic stroke is followed by blood-brain barrier (BBB) dysfunction and vasogenic brain edema. Structurally, tight junctions (TJs) between the endothelial cells play an important role in maintaining the integrity of the blood-brain barrier (BBB). IR injury is an early secondary injury leading to a non-specific, inflammatory response. Oxidative and metabolic stress following inflammation triggers secondary brain damage including BBB permeability and disruption of tight junction (TJ) integrity.
Our protocol presents an in vitro example of oxygen-glucose deprivation and reoxygenation (OGD-R) on rat brain endothelial cell TJ integrity and stress fiber formation. Currently, several experimental in vivo models are used to study the effects of IR injury; however they have several limitations, such as the technical challenges in performing surgeries, gene dependent molecular influences and difficulty in studying mechanistic relationships. However, in vitro models may aid in overcoming many of those limitations. The presented protocol can be used to study the various molecular mechanisms and mechanistic relationships to provide potential therapeutic strategies. However, the results of in vitro studies may differ from standard in vivo studies and should be interpreted with caution.

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 ...

MRI-guided dmPFC-rTMS as a Treatment for Treatment-resistant Major Depressive Disorder

Here we outline the protocol for magnetic resonance imaging (MRI) guided repetitive transcranial magnetic stimulation (rTMS) to the dorsal medial prefrontal cortex (dmPFC) in patients with major depressive disorder (MDD). Technicians used a neuronavigation system to process patient MRIs to generate a 3-dimensional head model. The head model was subsequently used to identify patient-specific stimulatory targets. The dmPFC was stimulated daily for 20 sessions. Stimulation intensity was titrated to address scalp pain associated with rTMS. Weekly assessments were conducted on the patients using the Hamilton Rating Scale for Depression (HamD17) and Beck Depression Index II (BDI-II). Treatment-resistant MDD patients achieved significant improvements on both HAMD and BDI-II. Of note, angled, double-cone coil rTMS at 120% resting motor threshold allows for optimal stimulation of deeper midline prefrontal regions, which results in a possible therapeutic application for MDD. One major limitation of the rTMS field is the heterogeneity of treatment parameters across studies, including duty cycle, number of pulses per session and intensity. Further work should be done to clarify the effect of stimulation parameters on outcome. Future dmPFC-rTMS work should include sham-controlled studies to confirm its clinical efficacy in MDD.

Here we outline the procedure for MRI-guided repetitive transcranial magnetic stimulation to the dorsomedial prefrontal cortex as an experimental treatment for major depressive disorder.

MRI引导dmPFC-rTMS治疗作为治疗难治性抑郁症

Here we outline the protocol for magnetic resonance imaging (MRI) guided repetitive transcranial magnetic stimulation (rTMS) to the dorsal medial ...

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by astrocytes, pericytes and brain endothelial cells (BECs). Here, we detail methods to assess BBB coverage using single cultures of immortalized human BECs, single cultures of primary mouse BECs, and a humanized triple culture model (BECs, astrocytes and pericytes) of the BBB. To highlight the applicability of the assays to disease states, we describe the effect of oligomeric amyloid-&#946; (oA&#946;), which is an important contributor to Alzheimer&#39;s disease (AD) progression, on BBB coverage. Further, we utilize the epidermal growth factor (EGF) to illuminate the drug screening potential of the techniques. Our results show that single and triple cultured BECs form meshwork-like structures under basal conditions, and that oA&#946; disrupts this cell meshwork formation and degenerates the preformed mesh structures, but EGF blocks this disruption. Thus, the techniques described are important for dissecting fundamental and disease-relevant processes that modulate BBB coverage.

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Maintaining blood-brain barrier coverage is key for the homeostasis of the central nervous system. This protocol describes in vitro techniques to delineate the fundamental and pathological processes that modulate blood-brain barrier coverage.

评估血脑屏障网状血管形成和破坏的体外测定

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by ...

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) ...