我们感谢马里娜·库舍里亚瓦、马克西姆·克里沃诺索夫、达里娜·亚库门科和叶夫根尼亚·贡查里克，他们支持我们的讨论，并为我们的讨论作出了有益的贡献。获得的数据是鲁斯兰·库茨博士论文的一部分。

下列程序是根据《赫尔辛基宣言》和《欧洲共同体实验动物使用准则》的建议进行的。内盖夫本古里安大学的动物护理委员会也批准了这些实验。
1. 为实验程序准备大鼠
<ol><li>选择成年雄性斯普拉格-道利大鼠，没有明示病理学，每只体重在300至350克之间。</li>
	<li>将所有大鼠在室温下保持在22°C，在实验前有12小时的光和暗周期。</li>
	<li>确保食物和水是可用的广告利比妥姆。</li>
	<li>执行下午 6：00 至下午 2：00 之间的所有程序.m.m。</li>
</ol>2. 准备大鼠做手术
<ol><li>用异氟化麻醉大鼠30分钟（感应4%，保养2%）和24%氧气（1.5升/分钟）。<ol>
<li>通过确保大鼠没有踏板取出反射来测试大鼠的麻醉水平。</li>
	</ol>
</li>
	<li>将 24 度导管插入尾静脉。 注：不执行血管化的尾部加热。<ol>
<li>把老鼠放在桌子上的姿势。用医用胶带把老鼠的四肢都贴上。</li>
	</ol>
</li>
	<li>手术前将探针放入大鼠直肠进行温度测量。</li>
	<li>在过程中，保持加热板以支持 37 °C 的核心体温。</li>
	<li>在老鼠的眼睛里加入软膏以保护。</li>
	<li>剃光手术区，消毒三种应用10%的波维酮碘，其次是70%的异丙醇。</li>
</ol>3. 右侧中脑动脉闭塞
注：MCAO采用修改后的技术，如先前描述的16、22、23，使用麦加里等人描述的仪器24和乌卢埃等人25。
<ol><li>用手术钳子解剖颈部腹中线的皮肤和表面筋膜，用弯曲的刀片剪刀。</li>
	<li>识别肌肉三角形，由ICA、外部胡萝卜动脉（ECA）和普通胡萝卜动脉（CCA）组成。</li>
	<li>小心地将右CCA和ICA与迷走神经与血管手术的微力分离。</li>
	<li>揭露正确的 CCA 和 ICA 。使用微型夹子或血管手术的特殊环状物阻止从CCA到ICA的血流。使用显微剪刀在ICA上切口（约1毫米），用于血管手术。</li>
	<li>直接通过ICA插入单丝导管（4-0尼龙），从右CCA的分叉点约18.5-19毫米进入威利斯圆，直到达到轻微的阻力，以遮挡MCA26。</li>
	<li>在ICA的分裂之上，在ICA周围展开。</li>
	<li>对于假操作的对照组，执行尼龙线的插入，而不是步骤3.5和3.616，22。</li>
	<li>通过腹膜注射管理5 mL的0.9%氯化钠。</li>
	<li>用缝合关闭伤口，将老鼠带到恢复区。 注意：麻醉结束后几分钟，老鼠会醒来，在笼子里独立移动。</li>
	<li>MCAO后23小时，通过针管27将2%的埃文斯蓝色盐水（4 mL/kg）23，26注入两个手术组的尾静脉。 注：这被用作血脑渗透度示踪器。允许循环60分钟。</li>
</ol>4. 确定梗塞区
<ol><li>测量IZ在24小时后，MCAO如前所述9，15，18，19，26。 注：体重减轻超过20%或出现癫痫发作或六肢瘫痪的老鼠被排除在实验之外。</li>
	<li>用20%的氧气和80%的二氧化碳代替受启发的气体混合物，直到大鼠停止自发呼吸，从而对大鼠实施安乐死。</li>
	<li>用剪刀和手术钳子在肋骨笼下的腹部壁上用5-6厘米的横向切口打开胸部。</li>
	<li>用剪刀和手术钳沿着肋骨笼的整个长度进行隔膜切口。</li>
	<li>小心地取代肺部，切开肋骨笼，直至左右两侧的锁骨28。</li>
	<li>通过心脏左心室注入200 mL的正常盐水。</li>
	<li>用剪刀刺穿或切入心脏右中庭。</li>
	<li>使用断头台进行斩首，并收集脑组织。</li>
	<li>使用虹膜剪刀，从前兆切割到两侧后头骨表面的分离边缘。</li>
	<li>将嗅灯泡、腹腔表面的神经连接和头骨的鼻孔表面与大脑分开。</li>
	<li>从头部取出大脑。</li>
	<li>通过创建 2 毫米厚的水平部分，使用 0.009" 不锈钢、未涂层、单边剃须刀刀片，生成 6 个脑片。</li>
	<li>在 0.05% TTC 中在 37 °C 下孵化 30 分钟。</li>
	<li>将脑组织放在显微镜幻灯片上，对分辨率为 1600x1600 dpi 的这 6 个脑片进行光学扫描（例如，参见补充 1）。</li>
	<li>使用通道混频器功能（图像 + 调整 + 通道混频器） 添加带有照片编辑器的蓝色滤镜（例如 Adobe Photoshop CS2），并将图像保存为 JPEG 文件格式。 注：应用蓝色滤镜后，图像将出现灰度。</li>
	<li>在 ImageJ 1.37v29、30中打开保存的图像。 注：此计算机程序使用阈值函数来隔离和计算黑色或白色的像素（见图 1）。</li>
	<li>对于图像的 6 个大脑切片中的每一个，使用主菜单中的"多边形选择"工具，选择并保存每个半球（右侧受伤的 ipsilateral 和左侧未受伤的反面）作为单独的图像文件。</li>
	<li>通过选择 Image > 调整 + 阈值，使用 ImageJ 软件主菜单中的自动阈值功能设置确定 IZ 的截止值，并测量单个大脑集每个半球的像素数。 注：宏可用于 ImageJ 软件中的此步骤（请参阅代码 补充 2）。 切断是确定哪些像素转换为白色，哪些转换为黑色取决于灰色阴影的关键参数（见 补充 3 和 补充 4 作为示例）。然后，ImageJ 比较白色和黑色像素以确定 IZ。根据染色协议和扫描仪设置，我们使用了 0.220 的恒定截止值。</li>
	<li>使用益普西拉特尔和反三边脑半球（RICH）方法13、23（见补充5中的示例）对组织肿胀进行IZ校正测量。 <img alt="Equation 1" src="/files/ftp_upload/61309/61309equ01.jpg"> 注：梗死大小被评估为反半球的百分比。</li>
</ol>5. 脑水肿的确定31
注：使用图像J 1.37v测量 BE32，33。
<ol><li>测量是 24 小时后， Mcao 。要计算 BE，请使用来自左右半球体积（单位）的数据。</li>
	<li>执行分辨率为 1600x1600 dpi 的光学扫描（例如，请参阅补充 1）。</li>
	<li>选择大脑半球，并设置用 ImageJ 1.37v 确定 BE 的截止点，如上文在 4.17-4.19 节中所述。</li>
	<li>将 BE 区域作为未受影响的反向半球标准区域的百分比，由 RICH 方法使用以下方程计算（见补充 5中的示例）23、34。 <img alt="Equation 2" src="/files/ftp_upload/61309/61309equ02v2.jpg"> 注：BE 的程度被评估为反半球的百分比。</li>
</ol>6. BBB 中断的确定
<ol><li>测量 MCAO 后 24 小时的 BBB 中断。</li>
	<li>将左右半球分成六个切片，每个切片放入微中心管中。</li>
	<li>根据计算4mL中1克脑组织50%三氯乙酸的三氯乙酸，将三氯乙酸中的每一片脑组织同质化。</li>
	<li>离心机在10，000 x g 20分钟。</li>
	<li>用 96% 乙醇稀释超纳坦液体 1：3。</li>
	<li>利用光谱学软件进行发光光谱测量，安装板材，并使用以下参数进行样品读取：荧光强度激发波长为620nm（带宽10nm），发射波长为680nm（带宽10nm）23，35：模组顶部;弗莱什25号;手动 100;摇晃1秒1毫米 注：使用620nm（带宽10nm）和680nm（带宽10nm）的激发波长。23，35
</li>
</ol>

作者没有什么可透露的。

本议定书的主要目标是证明对缺血损伤的三个主要参数的一致测量：IZ、BE和BBB渗透性。先前在这一领域的研究表明，有可能在同一样本中一起执行其中一个或两个参数。除了这种由三部分组成的方法提供的成本降低外，它还提供了一个更理想的生物伦理模型，限制必须操作并随后实施安乐死的动物数量。与所有病理学技术一样，该方法受到无法动态观察缺血损伤的限制。
在?...

全世界发病率和死亡率最常见的原因之一是中风。在全球范围内，缺血性中风占所有中风病例的68%，而在美国缺血性中风占中风病例的87%。1，2。据估计，中风的经济负担达到340亿美元，在美国2和450亿欧元在欧盟3。动物中风模型是研究其病理生理学，开发新的评价方法，并提出新的治疗方案4。
缺血性中风发生在一个主要脑动脉的闭塞，通常是中脑动脉或其分支之一5。因此，缺血性中风的模型历来涉及中脑动脉闭塞（MCAO）6，7，8，9，10，11，12。继 MCAO 之后， 神经损伤最常通过测量梗塞区 （IZ） 来评估，使用 2，3，5 三苯甲酸酯氯化物 （TTC） 染色方法13，脑水肿 （BE） 使用 干燥或计算半球体积14，15，16，和血脑屏障（BBB）渗透性光谱技术使用埃文斯蓝色染色17，18，19。
传统的 MCAO 方法在三个大脑测量中每个都使用单独的大脑集。对于大量的样本量，这导致大量的安乐死动物，增加了伦理和财务考虑。减轻这些成本的替代方法将涉及在一组后 MCAO 啮齿动物大脑中测量所有三个参数。
先前曾尝试测量同一大脑样本中的参数组合。同时免疫荧光染色方法20 以及其他分子和生化分析21 已描述后TTC染色在同一大脑样本。我们以前计算过脑半球的体积来评估脑水肿，并进行了TTC染色，以计算同一大脑集15的梗塞区。
在本协议中，我们提出了一种经过改进的 MCAO 技术，通过确定同一组啮齿动物大脑中的 IZ、BE 和 BBB 渗透性来测量缺血性脑损伤。IZ通过TTC染色测量，BE通过计算半球体积来测量，BBB渗透性通过光谱方法19获得。在此协议中，我们使用了经过修改的 MCAO 模型，基于将单滤导管直接插入和固定到内部胡萝卜动脉 （ICA） 和进一步阻断流向中脑动脉 （MCA）22的血液。与传统的MCAO方法16、22相比，这种修改后的方法表明死亡率和发病率有所下降。
这种新方法为测量 MCAO 之后的神经损伤提供了财务健全和道德模型。这种对缺血性脑损伤主要参数的评估将有助于全面研究其病理生理学。

<table>
	<tbody>
		<tr>
			<td>2 mL Syringe</td>
			<td>Braun</td>
			<td>4606027V</td>
			<td></td>
		</tr>
		<tr>
			<td>2% chlorhexidine in 70% alcohol solution</td>
			<td>Sigma-Aldrich</td>
			<td>500 cc</td>
			<td>Provides general antisepsis of the skin in the operatory field</td>
		</tr>
		<tr>
			<td>27 G Needle with Syringe</td>
			<td>Braun</td>
			<td>305620</td>
			<td></td>
		</tr>
		<tr>
			<td>3-0 Silk sutures</td>
			<td>Henry Schein</td>
			<td>1007842</td>
			<td></td>
		</tr>
		<tr>
			<td>4-0 Nylon suture</td>
			<td>4-00</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Brain &#38; Tissue Matrices</td>
			<td>Sigma-Aldrich</td>
			<td>15013</td>
			<td></td>
		</tr>
		<tr>
			<td>Cannula Venflon 22 G</td>
			<td>KD-FIX</td>
			<td>183603985447</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge Sigma 2-16P</td>
			<td>Sigma-Aldrich</td>
			<td>Sigma 2-16P</td>
			<td></td>
		</tr>
		<tr>
			<td>Compact Analytical Balances</td>
			<td>Sigma-Aldrich</td>
			<td>HR-AZ/HR-A</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital weighing scale</td>
			<td>Sigma-Aldrich</td>
			<td>Rs 4,000</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting scissors</td>
			<td>Sigma-Aldrich</td>
			<td>Z265969</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf pipette</td>
			<td>Sigma-Aldrich</td>
			<td>Z683884</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf tube</td>
			<td>Sigma-Aldrich</td>
			<td>EP0030119460</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence detector</td>
			<td>Tecan, M&#228;nnedorf Switzerland</td>
			<td>Model: Infinite 200 PRO multimode reader</td>
			<td>Optional.</td>
		</tr>
		<tr>
			<td>Fluorescence detector</td>
			<td>Molecular Devices LLC</td>
			<td>VWR cat. # 10822 512 SpectraMax Paradigm Multi Mode Microplate Reader Base Instrument</td>
			<td>Optional.</td>
		</tr>
		<tr>
			<td>Gauze sponges</td>
			<td>Fisher</td>
			<td>22-362-178</td>
			<td></td>
		</tr>
		<tr>
			<td>Heater with thermometer</td>
			<td>Heatingpad-1</td>
			<td>Model: HEATINGPAD-1/2</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemostatic microclips</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Horizon-XL</td>
			<td>Mennen Medical Ltd</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Infusion cuff</td>
			<td>ABN</td>
			<td>IC-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro forceps</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro scissors</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Multiset</td>
			<td>Teva Medical</td>
			<td>998702</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus BX 40 microscope</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Operating forceps</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Operating scissors</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Optical scanner</td>
			<td>Canon</td>
			<td>Cano Scan 4200F</td>
			<td>Resolution 3200 x 6400 dpi</td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>Sigma-Aldrich</td>
			<td>P5606</td>
			<td></td>
		</tr>
		<tr>
			<td>Purina Chow</td>
			<td>Purina</td>
			<td>5001</td>
			<td>Rodent laboratory chow given to rats, mice and hamster is a life-cycle nutrition that has been used in biomedical research for over 5 decades. Provided to rats ad libitum in this experiment.</td>
		</tr>
		<tr>
			<td>Rat cages</td>
			<td>Techniplast</td>
			<td>2000P</td>
			<td>Conventional housing for rodents. Cages were used for housing rats throughout the experiment</td>
		</tr>
		<tr>
			<td>Scalpel blades #11</td>
			<td>Sigma-Aldrich</td>
			<td>S2771</td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Adobe Photoshop CS2 for Windows</td>
			<td>Adobe</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ 1.37v</td>
			<td>NIH</td>
			<td></td>
			<td>The source code is freely available. The author, Wayne Rasband (wayne@codon.nih.gov), is at the Research Services Branch, National Institute of Mental Health, Bethesda, Maryland, USA</td>
		</tr>
		<tr>
			<td>Office 365 ProPlus</td>
			<td>Microsoft</td>
			<td>-</td>
			<td>Microsoft Office Excel</td>
		</tr>
		<tr>
			<td>Windows 10</td>
			<td>Microsoft</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2,3,5-Triphenyltetrazolium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>298-96-4</td>
			<td></td>
		</tr>
		<tr>
			<td>50% trichloroacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>76-03-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 96 %</td>
			<td>Romical</td>
			<td></td>
			<td>Flammable liquid</td>
		</tr>
		<tr>
			<td>Evans blue 2%</td>
			<td>Sigma-Aldrich</td>
			<td>314-13-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane, USP 100%</td>
			<td>Piramamal Critical Care, Inc</td>
			<td>NDC 66794-017</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring post-stroke cerebral edema, infarct zone and blood-brain barrier breakdown in a single set of rodent brain samples

全世界发病率和死亡率最常见的原因之一是缺血性中风。从历史上看，用于刺激缺血性中风的动物模型涉及中脑动脉闭塞（MCAO）。梗塞区、脑水肿和血脑屏障 （BBB） 故障被测量为反映 MCAO 后脑损伤程度的参数。这种方法的一个重大限制是，这些测量通常在不同的大鼠大脑样本中获得，由于大量大鼠需要安乐死以达到适当的样本大小，因此会导致伦理和经济负担。在这里，我们提出了一种方法，通过测量同一组大鼠大脑中的梗塞区、脑水肿和BBB渗透率，准确评估MCAO之后的脑损伤。这种新技术为评估中风的病理生理学提供了更有效的方法。

梗塞区测量
一项独立样本T测试表明，与16只假手术大鼠相比，19只接受永久性MCAO的老鼠的大脑梗塞体积显著增加（MCAO=7.49%，±3.57对。 香=0.31%±1.9，t（28.49）=7.56，p<0.01（见图2A）。 数据表示为反半球± SD 的平均百分比。
脑水肿测量
一项独立样本T测试表明，与16只假手术大...

Watch this Scientific Journal Video about Measuring Post-Stroke Cerebral Edema, Infarct Zone and Blood-Brain Barrier Breakdown in a Single Set of Rodent Brain Samples at JoVE.com

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

此协议描述了在同一组啮齿动物脑样本上测量缺血性脑损伤三个最重要的参数的新技术。仅使用一个大脑样本在道德和经济成本方面非常有利。

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 ...

measuring-post-stroke-cerebral-edema-infarct-zone-blood-brain-barrier

Research

JoVE Journal

Neuroscience

7.8K 次观看.  Ben-Gurion University of the Negev. 此协议描述了在同一组啮齿动物脑样本上测量缺血性脑损伤三个最重要的参数的新技术。仅使用一个大脑样本在道德和经济成本方面非常有利。

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

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

使用超声波血脑屏障破坏和锰增强MRI的脑功能成像

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

科研

神经科学

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 ...

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 ...

Effects of Blast-induced Neurotrauma on Pressurized Rodent Middle Cerebral Arteries

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral vascular effects. Impact (i.e., non-blast) traumatic brain injury (TBI) is known to decrease pressure autoregulation in the cerebral vasculature in both humans and experimental animals. The hypothesis that blast-induced traumatic brain injury (bTBI), like impact TBI, results in impaired cerebral vascular reactivity was tested by measuring myogenic dilatory responses to reduced intravascular pressure in rodent middle cerebral arterial (MCA) segments from rats subjected to mild bTBI using an Advanced Blast Simulator (ABS) shock tube. Adult, male Sprague-Dawley rats were anesthetized, intubated, ventilated and prepared for Sham bTBI (identical manipulation and anesthesia except for blast injury) or mild bTBI. Rats were randomly assigned to receive Sham bTBI or mild bTBI followed by sacrifice 30 or 60 min post-injury. Immediately after bTBI, righting reflex (RR) suppression times were assessed, euthanasia at the time points post-injury was completed, the brain was harvested and the individual MCA segments were collected, mounted and pressurized. As the intraluminal pressure perfused through the arterial segments was reduced in 20 mmHg increments from 100 to 20 mmHg, MCA diameters were measured and recorded. With decreasing intraluminal pressure, MCA diameters steadily increased significantly above baseline in the Sham bTBI groups while MCA dilator responses were significantly reduced (p &#60; 0.05) in both bTBI groups as evidenced by the impaired, smaller MCA diameters recorded for the bTBI groups. In addition, RR suppression in the bTBI groups was significantly (p &#60; 0.05) higher than in the Sham bTBI groups. MCA&#39;s collected from the Sham bTBI groups exhibited typical vasodilatory properties to decreases in intraluminal pressure while MCA&#39;s collected following bTBI exhibited significantly impaired myogenic vasodilatory responses to reduced pressure that persisted for at least 60 min after bTBI.

Here, we present a protocol to describe methods for ex vivo vascular reactivity determination following a primary blast traumatic brain injury (bTBI) using isolated, pressurized, rodent middle cerebral arterial (MCA) segments. bTBI induction is accomplished using a shock tube, also known as an Advanced Blast Simulator (ABS) device.

爆炸性神经损伤对压生啮鼠中脑动脉的影响

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral ...

Focused Ultrasound Induced Blood-Brain Barrier Opening for Targeting Brain Structures and Evaluating Chemogenetic Neuromodulation

Acoustically Targeted Chemogenetics (ATAC) allows for the noninvasive control of specific neural circuits. ATAC achieves such control through a combination of focused ultrasound (FUS) induced blood-brain barrier opening (FUS-BBBO), gene delivery with adeno-associated viral (AAV) vectors, and activation of cellular signaling with engineered, chemogenetic, protein receptors and their cognate ligands. With ATAC, it is possible to transduce both large and small brain regions with millimeter precision using a single noninvasive ultrasound application. This transduction can later allow for a long-term, noninvasive, device-free neuromodulation in freely moving animals using a drug. Since FUS-BBBO, AAVs, and chemogenetics have been used in multiple animals, ATAC should also be scalable for the use in other animal species. This paper expands upon a previously published protocol and outlines how to optimize the gene delivery with FUS-BBBO to small brain regions with MRI-guidance but without a need for a complicated MRI-compatible FUS device. The protocol, also, describes the design of mouse targeting and restraint components that can be 3D-printed by any lab and can be easily modified for different species or custom equipment. To aid reproducibility, the protocol describes in detail how the microbubbles, AAVs, and venipuncture were used in ATAC development. Finally, an example data is shown to guide the preliminary investigations of studies utilizing ATAC.

This protocol delineates steps necessary for the gene delivery through focused ultrasound blood brain barrier (BBB) opening, evaluation of the resulting gene expression, and measurement of neuromodulation activity of chemogenetic receptors through histological tests.

聚焦超声诱导血脑屏障打开，用于靶向大脑结构和评估化学遗传神经调控

Acoustically Targeted Chemogenetics (ATAC) allows for the noninvasive control of specific neural circuits. ATAC achieves such control through a ...

跨内皮抗性测量以评估细胞屏障完整性

Barrier Functional Integrity Recording on bEnd.3 Vascular Endothelial Cells via Transendothelial Electrical Resistance Detection

The blood-brain barrier (BBB) is a dynamic physiological structure composed of microvascular endothelial cells, astrocytes, and pericytes. By coordinating the interaction between restricted transit of harmful substances, nutrient absorption, and metabolite clearance in the brain, the BBB is essential in preserving central nervous system homeostasis. Building in vitro models of the BBB is a valuable tool for exploring the pathophysiology of neurological disorders and creating pharmacological treatments. This study describes a procedure for creating an in vitro monolayer BBB cell model by seeding bEnd.3 cells into the upper chamber of a 24-well plate. To assess the integrity of cell barrier function, the conventional epithelial cell voltmeter was used to record the transmembrane electrical resistance of normal cells and CoCl2-induced hypoxic cells in real-time. We anticipate that the above experiments will provide effective ideas for the creation of in vitro models of BBB and drugs to treat disorders of central nervous system diseases.

作者聚焦：TEER 检测在评估细胞屏障完整性中的应用 

This protocol describes a dependable and efficient in vitro model of the brain blood barrier. The method uses mouse cerebral vascular endothelial cells bEnd.3 and measures transmembrane electrical resistance.

通过跨内皮电阻检测对bEnd.3血管内皮细胞进行屏障功能完整性记录

The blood-brain barrier (BBB) is a dynamic physiological structure composed of microvascular endothelial cells, astrocytes, and pericytes. By coordinating ...

A 3D Blood-Brain Barrier Model Derived from Human Pluripotent Stem-Cells

Reconstruction of the Blood-Brain Barrier In Vitro to Model and Therapeutically Target Neurological Disease

The blood-brain barrier (BBB) is a key physiological component of the central nervous system (CNS), maintaining nutrients, clearing waste, and protecting the brain from pathogens. The inherent barrier properties of the BBB pose a challenge for therapeutic drug delivery into the CNS to treat neurological diseases. Impaired BBB function has been related to neurological disease. Cerebral amyloid angiopathy (CAA), the deposition of amyloid in the cerebral vasculature leading to a compromised BBB, is a co-morbidity in most cases of Alzheimer&#39;s disease (AD), suggesting that BBB dysfunction or breakdown may be involved in neurodegeneration. Due to limited access to human BBB tissue, the mechanisms that contribute to proper BBB function and BBB degeneration remain unknown. To address these limitations, we have developed a human pluripotent stem cell-derived BBB (iBBB) by incorporating endothelial cells, pericytes, and astrocytes in a 3D matrix. The iBBB self-assembles to recapitulate the anatomy and cellular interactions present in the BBB. Seeding iBBBs with amyloid captures key aspects of CAA. Additionally, the iBBB offers a flexible platform to modulate genetic and environmental factors implicated in cerebrovascular disease and neurodegeneration, to investigate how genetics and lifestyle affect disease risk. Finally, the iBBB can be used for drug screening and medicinal chemistry studies to optimize therapeutic&#160;delivery to the CNS. In this protocol, we describe the differentiation of the three types of cells (endothelial cells, pericytes, and astrocytes) arising from human pluripotent stem cells, how to assemble the differentiated cells into the iBBB, and how to model CAA in vitro using exogenous amyloid. This model overcomes the challenge of studying live human brain tissue with a system that has both biological fidelity and experimental flexibility, and enables the interrogation of the human BBB and its role in neurodegeneration.

Author Spotlight: A Personalized Approach Towards Investigating Alzheimer's Disease Using an In Vitro Blood-Brain Barrier Model 

The blood-brain barrier (BBB) has a crucial role in sustaining a stable and healthy brain environment. BBB dysfunction is associated with many neurological diseases. We have developed a 3D, stem-cell-derived model of the BBB to investigate cerebrovascular pathology, BBB integrity, and how the BBB is altered by genetics and disease.

体外血脑屏障重建，模拟和治疗神经系统疾病

The blood-brain barrier (BBB) is a key physiological component of the central nervous system (CNS), maintaining nutrients, clearing waste, and protecting ...