这项工作得到了 "基金会 Neuromed" 的支持, 由意大利卫生部 "74年2月 Corrente" 资助科诺诺夫

所有动物程序均由 IRCCS Neuromed 动物保育审查委员会和 #34 批准; 依 Superiore Sanit 和 #224; #34; (许可证编号: 1163/2015-PR), 并按照欧盟指令2010的指导方针/63/欧洲动物实验. 
 1. 注射到颈静脉内的 FITC-白蛋白溶液的制备 注意: 所有实验都是在清单 (11 周龄) HD R6/2 小鼠和年龄和性别匹配的野生型 (重量) 控制中进行的。窝. 
<ol> <li> 在磷酸缓冲盐水 (PBS) 中溶解 FITC-白蛋白粉末 (参见 材料表 ), 在10毫克/毫升。
	注: 为获得最佳效果, 应为每个标签反应准备新的. </li>
</ol> 2。外科手术中使用的设备的准备工作 <ol> <li> 放置以前的蒸压的外科仪器 (请参见 材料表 )。
	<li> 用酒精 (70% 乙醇溶液) 清洗手术台。准备手术区 (45 厘米 x 45 厘米) 与外科无菌一次性毛巾悬垂和设置操作显微镜. </li>
</ol> 3。FITC-白蛋白输注到颈静脉的手术方法 <ol> <li> 记录麻醉时的鼠标体重. </li>
	<li> 麻醉鼠腹腔注射 (i. p) 的适当剂量氯胺酮 (100 毫克/千克)-嗪 (10 毫克/千克) 溶液. </li>
	<li> 在 Walantus et al. 中, 通过轻柔的脚趾掐缩反应来监测动物镇静反应, 如下所示 11 , 12 . </li> <li> 将麻醉鼠标置于正面仰卧位置, 并用胶带固定在手术台上的四条腿和尾部. </li>
	<li> 在鼠标安全后, 小心地用电动剃须刀将颈部适当的手术边缘剃掉 (参见 材料表 ), 并用聚维酮碘溶液和70% 乙醇对其进行消毒. </li>
	<li> 用棉签将裸露的剃光表面烘干. </li>
	<li> 轻轻地拉起剃光的皮肤, 用手术刀做一个1.5 厘米长的纵向切口, 在锁骨线上, 从胸部的中段开始, 延伸到颈部以下. </li>
	<li> 在显微镜观察 (5X 放大倍率) 下, 仔细地将结缔组织与镊子分开, 以暴露颈外静脉. </li>
	<li> 确保在右侧和颈静脉左侧有足够的空间, 以方便插入30和 #189; G 针用于 FITC-白蛋白溶液的注入. </li>
	<li> 通过使用30和 #189, 静脉注射 (右颈静脉) (3 分钟以上) 与100和 #181; L 10 毫升/千克 FITC-白蛋白; G 针. </li>
	<li> 15 分钟后, 安乐将鼠标斩首, 迅速从头骨上移除先前描述的大脑 13 , 并将其浸入15分钟, 至少10x 的大脑本身的 pre-chilled 戊. </li>
	<li> 将戊冷冻的大脑移动到 pre-chilled 管中, 并将其存储到-80 和 #176; C 冷冻直到组织学切片. </li>
</ol> 4。FITC-白蛋白注入脑组织切片的研究 <ol> <li> 在最佳切削温度 (OCT) 复合物中嵌入冷冻大脑 (见 材料表 ) 在低温切片之前. </li>
	<li> 按顺序将大脑切成20和 #181; m 厚部分, 低温 (请参阅 材料表 ), 并将其安装到显微镜玻璃幻灯片上. </li>
	<li> 使用特定的 anti-laminin 抗体 (见材料表) 执行 FITC-白蛋白/层粘连蛋白 co-staining, 如前所述 6 。
	<ol> <li> 简要地, 在夜间与主抗体 anti-laminin (pAb 1:1000) 孵育切片, 并进行适当的二级抗体共轭 Cy3. </li>
		<li> 片幻灯片, 安装介质包含4和 #39;, 6-diamidino-2-吲 (DAPI) (吸收峰值 = 360 nm; 发射峰值 = 460 nm (请参阅 材料表 ) </li>
		<li> 使幻灯片在4和 #176 中通宵晾干; C 在黑暗中. </li>
		<li> 在荧光显微镜下观察荧光染色 (参见 材料表 ), 在20X 倍放大器上装有两个 FITC (吸收峰值 = 495 nm; 发射峰值 = 525 nm) 和 Cy3 滤镜 (吸收峰值 = 550 nm; 发射峰值 = 570 nm)。
		<ol> <li> 在染色后立即查看样品, 用指甲油固定在角上的片, 并在显微镜下检查, 如上图 (4.3.3). </li>
		</ol> </li> </ol> </li> </ol> 5。设置荧光显微镜和彩色图像采集 <ol> <li> 设置荧光显微镜如下. 
	<ol> <li> 在使用前10分钟打开萤光灯。打开显微镜、连接的计算机和数码相机。运行图像分析软件 (请参见 材料表 ). </li>
		<li> 使用适当的目标来优化信号收集和空间分辨率, 并选择适当的筛选器. </li>
	</ol> </li> <li> 获取彩色图像 <ol> <li> 在图像分析软件中选择 [实时] 命令 (参见 材料表 ), 并为每个滤镜设置最佳光照参数. </li>
		<li> 选择 [冻结] 命令以获得单通道彩色图像并添加缩放栏 (#39; 编辑和 #62; 刻录缩放和 #39;) </li>
		<li> 保存 #39 中的每个图像; tiff 和 #39; 格式. </li>
		<li> 通过选择和 #39; 文件和 #62; 合并通道和 #39, 将通道合并到单个图像中; 并将其保存在 </li>
		<li> tiff 和 #39; 格式 </li>
		<li> 每个脑切片 (每张幻灯片6节) 获取至少四二十四位彩色图片 (2200 和 #181; m x 3400 和 #181; m). </li>
	</ol> </li> </ol> 6。FITC-白蛋白外渗的评价 <ol> <li> 通过使用自由可用的 ImageJ 来分析每个通道的荧光强度 14 , 15 . </li>
	<li> 通过每个图像的总 CY3-Laminin 荧光强度使 FITC 白蛋白的总荧光信号强度正常化, 并将血管外的淤血白蛋白报告为绿色荧光强度. </li>
</ol>

<ol>
	<li>Obermeier, B., Verma, A., Ransohoff, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+blood-brain+barrier.">The blood-brain barrier.</a> Handb Clin Neurol. 133, 39-59 (2016).</li><li>Serlin, Y., Shelef, I., Knyazer, B., Friedman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+and+physiology+of+the+blood-brain+barrier.">Anatomy and physiology of the blood-brain barrier.</a> Semin Cell Dev Biol. 38, 2-6 (2015).</li><li>Moretti, R., 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=Blood-brain+barrier+dysfunction+in+disorders+of+the+developing+brain.">Blood-brain barrier dysfunction in disorders of the developing brain.</a> Front Neurosci. 9, 40 (2015).</li><li>Zhao, Z., Nelson, A. R., Betsholtz, C., Zlokovic, B. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+Dysfunction+of+the+Blood-Brain+Barrier.">Establishment and Dysfunction of the Blood-Brain Barrier.</a> Cell. 163 (5), 1064-1078 (2015).</li><li>Drouin-Ouellet, J., 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=Cerebrovascular+and+blood-brain+barrier+impairments+in+Huntington's+disease:+Potential+implications+for+its+pathophysiology.">Cerebrovascular and blood-brain barrier impairments in Huntington's disease: Potential implications for its pathophysiology.</a> Ann Neurol. 78 (2), 160-177 (2015).</li><li>Di Pardo, A., 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=Impairment+of+blood-brain+barrier+is+an+early+event+in+R6/2+mouse+model+of+Huntington+Disease.">Impairment of blood-brain barrier is an early event in R6/2 mouse model of Huntington Disease.</a> Sci Rep. 7, 41316 (2017).</li><li>Lecler, A., Fournier, L., Diard-Detoeuf, C., Balvay, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood-Brain+Barrier+Leakage+in+Early+Alzheimer+Disease.">Blood-Brain Barrier Leakage in Early Alzheimer Disease.</a> Radiology. 282 (3), 923-925 (2017).</li><li>van de Haar, H. J., 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=Blood-Brain+Barrier+Leakage+in+Patients+with+Early+Alzheimer+Disease.">Blood-Brain Barrier Leakage in Patients with Early Alzheimer Disease.</a> Radiology. 282 (2), 615 (2017).</li><li>Fernandez-Lopez, D., 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=Blood-brain+barrier+permeability+is+increased+after+acute+adult+stroke+but+not+neonatal+stroke+in+the+rat.">Blood-brain barrier permeability is increased after acute adult stroke but not neonatal stroke in the rat.</a> J Neurosci. 32 (28), 9588-9600 (2012).</li><li>Yang, Y., Rosenberg, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood-brain+barrier+breakdown+in+acute+and+chronic+cerebrovascular+disease.">Blood-brain barrier breakdown in acute and chronic cerebrovascular disease.</a> Stroke. 42 (11), 3323-3328 (2011).</li><li>Walantus, W., Castaneda, D., Elias, L., Kriegstein, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+utero+intraventricular+injection+and+electroporation+of+E15+mouse+embryos.">In utero intraventricular injection and electroporation of E15 mouse embryos.</a> J Vis Exp. (6), e239 (2007).</li><li>Szot, G. L., Koudria, P., Bluestone, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+pancreatic+islets+into+the+kidney+capsule+of+diabetic+mice.">Transplantation of pancreatic islets into the kidney capsule of diabetic mice.</a> J Vis Exp. (9), e404 (2007).</li><li>Gage, G. J., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+animal+perfusion+fixation+for+rodents.">Whole animal perfusion fixation for rodents.</a> J Vis Exp. (65), (2012).</li><li>McCloy, R. A., 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=Partial+inhibition+of+Cdk1+in+G+2+phase+overrides+the+SAC+and+decouples+mitotic+events.">Partial inhibition of Cdk1 in G 2 phase overrides the SAC and decouples mitotic events.</a> Cell Cycle. 13 (9), 1400-1412 (2014).</li><li>Burgess, A., 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=Loss+of+human+Greatwall+results+in+G2+arrest+and+multiple+mitotic+defects+due+to+deregulation+of+the+cyclin+B-Cdc2/PP2A+balance.">Loss of human Greatwall results in G2 arrest and multiple mitotic defects due to deregulation of the cyclin B-Cdc2/PP2A balance.</a> Proc Natl Acad Sci U S A. 107 (28), 12564-12569 (2010).</li><li>Krueger, M., Hartig, W., Reichenbach, A., Bechmann, I., Michalski, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood-brain+barrier+breakdown+after+embolic+stroke+in+rats+occurs+without+ultrastructural+evidence+for+disrupting+tight+junctions.">Blood-brain barrier breakdown after embolic stroke in rats occurs without ultrastructural evidence for disrupting tight junctions.</a> PLoS One. 8 (2), e56419 (2013).</li><li>Hirano, A., Kawanami, T., Llena, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+microscopy+of+the+blood-brain+barrier+in+disease.">Electron microscopy of the blood-brain barrier in disease.</a> Microsc Res Tech. 27 (6), 543-556 (1994).</li></ol>

作者没有利益冲突披露

我们在这里描述的技术主要是用来检测脑疾病状态下的 BBB 渗漏。BBB 功能障碍是越来越多的关注, 作为一个常见的特点, 不同的神经系统疾病4。我们以前使用这种方法来描述在一个罕见的神经退行性疾病的小鼠模型, 如 HD6早期紊乱的 BBB 完整性。
该方法利用了程序的相对简单性和有效性, 提供了准确、可靠的结果。它代表了一个高度敏感的?...

An erratum was issued for: <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/56389/assessment-blood-brain-barrier-permeability-intravenous-infusion-fitc">Assessment of Blood-brain Barrier Permeability by Intravenous Infusion of FITC-labeled Albumin in a Mouse Model of Neurodegenerative Disease</a>. One of the author&#39;s names was corrected from:
Maglione Vittorio
to:
Vittorio Maglione

An erratum was issued for: <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/56389/assessment-blood-brain-barrier-permeability-intravenous-infusion-fitc">Assessment of Blood-brain Barrier Permeability by Intravenous Infusion of FITC-labeled Albumin in a Mouse Model of Neurodegenerative Disease</a>. One of the author's names was corrected.

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

中枢神经系统内稳态是神经元细胞正常沟通和功能的先决条件。中枢神经系统的实质是由内皮血脑屏障 (BBB) 与周围紧密封闭的, 它代表着外周血和脑的接口, 在这两个区之间的相声中起着举足轻重的作用1 ,2。血脑屏障是一种复杂的动态三维结构, 主要由专门的微血管内皮细胞 (ECs) 通过细胞间连接-紧密接合 (TJs) 和周, 神经元结尾和星形胶质细胞脚处理1,2。
在生理条件下, 完整的血脑屏障的极低通透性确保了对营养物质和其他分子进出大脑的严格调节, 并为中枢神经系统提供了独特的保护, 使其不会发生可能影响神经活动和反对可能的周边侮辱的血液的构成1,2,3。
长期以来, 对 BBB 完整性及其增强的渗透性的破坏已经成为许多神经和神经退行性疾病的关键特征4包括亨廷顿氏病 (HD)5,6, 但是, 这种功能失调是一种致病现象还是在病程中繁殖事件尚不清楚。血脑屏障破裂的时间也仍然难以捉摸, 然而, 我们组和其他人的新证据表明, 扰乱 bbb 完整性并不代表疾病进展的晚期事件, 而是早期的步骤6,7,8, 可能会产生长期后果。
考虑到这一点, 重要的是要早熟揭示 BBB 故障在神经, 以制定战略有用的预测疾病进展和脑损伤, 并制定替代和更有针对性的干预, 能够成功减轻这种破坏的临床后果。因此, 对脑屏障损害的可靠影像学研究, 在大脑疾病的实验和临床管理中具有重要意义。
本文介绍了利用高分子量荧光素异硫氰酸酯标记白蛋白 (FITC-白蛋白) 在 HD 小鼠模型中评价 BBB 通透性的一种成功而简单的方法。FITC-白蛋白的外渗, 通常不能越过屏障, 进入脑实质被测量为 BBB 渗漏的一个指标。该技术易于适应大鼠和其他病理条件的特点, cerebrovasculature 损害9,10。

<table>
	<tbody>
		<tr>
			<td>Albumin-fluorescin isothiocyanate conjiugate</td>
			<td>SIGMA</td>
			<td>A9771-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>pAb anti-Laminin</td>
			<td>Novus Biologicals</td>
			<td>NB300-144</td>
			<td></td>
		</tr>
		<tr>
			<td>CY3 anti-rabbit made in goat</td>
			<td>MILLIPORE</td>
			<td>AP132</td>
			<td></td>
		</tr>
		<tr>
			<td>SUPERFROST PLUS</td>
			<td>Thermo Scientific</td>
			<td>J1800AMNZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover Slips 24 X 50 mm</td>
			<td>Thermo Scientific (DIAPATH)</td>
			<td>61050</td>
			<td></td>
		</tr>
		<tr>
			<td>Kilik Optimal Cutting Temperature (OCT) compound</td>
			<td>Bio Optica</td>
			<td>05-9801</td>
			<td></td>
		</tr>
		<tr>
			<td>VECTASHIELD Mounting Media</td>
			<td>VECTOR</td>
			<td>H-1500</td>
			<td>Mounting media with DAPI</td>
		</tr>
		<tr>
			<td>iNSu/Light Insulin Disposible Syringe</td>
			<td>RAYS Health &#38;Safety</td>
			<td>INS1ML26G13</td>
			<td></td>
		</tr>
		<tr>
			<td>30G 1/2&#34;</td>
			<td>BD Microlance</td>
			<td>304000</td>
			<td>Needle for Insulin disposible Syringe</td>
		</tr>
		<tr>
			<td>Scalpel Handle</td>
			<td>F.S.T.</td>
			<td>91003-12</td>
			<td></td>
		</tr>
		<tr>
			<td>#22 Disposable Scalpel blads</td>
			<td>F.S.T.</td>
			<td>10022-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Iris scissors 10.5 cm</td>
			<td>F.S.T.</td>
			<td>14094-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont Forceps #5745 45&#176; 0.10 x 0.06 mm</td>
			<td>F.S.T.</td>
			<td>11251-35</td>
			<td></td>
		</tr>
		<tr>
			<td>Graefe Forceps 10 cm</td>
			<td>F.S.T.</td>
			<td>11051-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont Forceps #5 0.1 X 0.06 mm</td>
			<td>F.S.T.</td>
			<td>11251-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Medical patch</td>
			<td>Medicalis</td>
			<td>34788</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile disposable towel drape</td>
			<td>Dispotech</td>
			<td>TVO50VE</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereoscopic Microscope</td>
			<td>NIKON</td>
			<td>SMZ 745 T</td>
			<td></td>
		</tr>
		<tr>
			<td>Optic Illuminator LED light (C-FLED2)</td>
			<td>NIKON</td>
			<td>1003167</td>
			<td>Optic Illuminator for Stereoscopic Micrscope</td>
		</tr>
		<tr>
			<td>Eclipse Ni-U Microscope</td>
			<td>Nikon</td>
			<td>932162</td>
			<td>Epifluorescence Microscope</td>
		</tr>
		<tr>
			<td>Microscope digital Camera</td>
			<td>Nikon</td>
			<td>DS-Ri2</td>
			<td>Microscope camera</td>
		</tr>
		<tr>
			<td>Intenslight</td>
			<td>Nikon</td>
			<td>C-HGFI</td>
			<td>Microscope lamp</td>
		</tr>
		<tr>
			<td>NIS-Elements 64 bit</td>
			<td>Nikon</td>
			<td>AR 4.40.00</td>
			<td>Analysis Software</td>
		</tr>
		<tr>
			<td>Electric Razor</td>
			<td>Gemei</td>
			<td>GM-3007</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessment of blood-brain barrier permeability by intravenous infusion of fitc-labeled albumin in a mouse model of neurodegenerative disease

破坏血脑屏障 (BBB) 的完整性是一个常见的特点, 不同的神经和神经退行性疾病。虽然扰动 bbb 稳态和脑疾病的发病机制之间的相互作用需要进一步的研究, 开发和验证可靠的程序, 以准确地检测 bbb 改变可能是至关重要的, 并代表了一个有用的工具潜在的预测疾病进展和制定有针对性的治疗策略。
在这里, 我们提出了一个简单和有效的程序, 评估脑屏障渗漏的神经退行性的情况, 如发生在一个前临床小鼠亨廷顿疾病模型, 其中的缺陷, 血 bbb 的渗透性是明确可探测早熟这种疾病。具体来说, 高分子量的荧光素异硫氰酸酯标记 (FITC)-白蛋白, 只有当后者被削弱时才能够穿过 BBB, 在血管或实质区内被剧烈注入小鼠颈静脉并分布然后由荧光显微镜测定。
绿色荧光-白蛋白在脑实质中的积累是异常 BBB 通透性的一个指标, 当定量使用图像 J 处理软件时, 报告为绿色荧光强度。

适当的 FITC-白蛋白注入颈静脉的结果在绿色荧光示踪剂从血液进入大脑 parenchymawhen BBB 是危及6。在生理条件下, 注入的荧光白蛋白的分布仅限于脑血管内, 周围血管区和/或脑实质的信号都是可探测的 (图 1A, 显微顶)。相反, 当血脑屏障在神经病理环境下被破坏时, FITC-白蛋白在血管空间和大脑实质 (图 1A, 底部显?...

Watch this Scientific Journal Video about Assessment of Blood-brain Barrier Permeability by Intravenous Infusion of FITC-labeled Albumin in a Mouse Model of Neurodegenerative Disease at JoVE.com

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

在这项研究中, 我们提出了一个简单和有效的程序来评估在神经退行性条件下血脑屏障的破坏。为了达到我们的目标, 我们在小鼠颈静脉内注入了高分子荧光素异硫氰酸酯标记 (FITC)-白蛋白, 并通过荧光显微镜将其渗漏到脑实质中。

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 ...

assessment-blood-brain-barrier-permeability-intravenous-infusion-fitc

Research

JoVE Journal

Neuroscience

9.8K 次观看.  IRCCS Neuromed. 在这项研究中, 我们提出了一个简单和有效的程序来评估在神经退行性条件下血脑屏障的破坏。为了达到我们的目标, 我们在小鼠颈静脉内注入了高分子荧光素异硫氰酸酯标记 (FITC)-白蛋白, 并通过荧光显微镜将其渗漏到脑实质中。

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

Methods for Intravenous Self Administration in a Mouse Model

Animal models have been developed to study the reinforcing effects of drugs, including the intravenous self-administration (IVSA) paradigm. The advantages of using an IVSA paradigm to study the reinforcing properties of drugs of abuse such as cocaine include the fact that the drug is self-administered instead of experimenter-administered, the schedule of reinforcement can be altered, and accurate measurement of the quantities of drug consumed as well as the timing and pattern of IV injections can be obtained. Furthermore, the intravenous route of administration avoids potential confounds related to first pass metabolism or taste, and produces rapid increases in blood and brain drug levels. As outlined in this video, intravenous self-administration can be obtained without prior food restriction or prior drug training following careful catheter placement during surgery and meticulous daily catheter flushing and maintenance. Experimental procedures outlined in this paper include a description of animal housing and acclimation methods, operant training using sweetened milk solutions, and catheter implantation surgery.

The intravenous self-administration (IVSA) paradigm is considered to be the gold standard in examining the reinforcing properties of drugs of abuse in rodents. This manuscript outlines the experimental procedures and surgical techniques necessary to obtain reliable IVSA data. In particular, meticulous catheter implantation and maintenance are highlighted.

在小鼠模型中静脉自我管理的方法

Animal models have been developed to study the reinforcing effects of drugs, including the intravenous self-administration (IVSA) paradigm. The advantages ...

科研

神经科学

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The blood-brain barrier (BBB) consisting of EC, astrocyte end-feet, pericytes and the basal membrane is responsible for the protection and homeostasis of the brain parenchyma. In vitro BBB models are common tools to study the structure and function of the BBB at the cellular level. A considerable number of different in vitro BBB models have been established for research in different laboratories to date. Usually, the cells are obtained from bovine, porcine, rat or mouse brain tissue (discussed in detail in the review by Wilhelm et al. 1). Human tissue samples are available only in a restricted number of laboratories or companies 2,3. While primary cell preparations are time consuming and the EC cultures can differ from batch to batch, the establishment of immortalized EC lines is the focus of scientific interest.
Here, we present a method for establishing an immortalized brain microvascular EC line from neonatal mouse brain. We describe the procedure step-by-step listing the reagents and solutions used. The method established by our lab allows the isolation of a homogenous immortalized endothelial cell line within four to five weeks. The brain microvascular endothelial cell lines termed cEND 4 (from cerebral cortex) and cerebEND 5 (from cerebellar cortex), were isolated according to this procedure in the F&ouml;rster laboratory and have been effectively used for explanation of different physiological and pathological processes at the BBB. Using cEND and cerebEND we have demonstrated that these cells respond to glucocorticoid- 4,6-9 and estrogen-treatment 10 as well as to pro-infammatory mediators, such as TNFalpha 5,8. Moreover, we have studied the pathology of multiple sclerosis 11 and hypoxia 12,13 on the EC-level. The cEND and cerebEND lines can be considered as a good tool for studying the structure and function of the BBB, cellular responses of ECs to different stimuli or interaction of the EC with lymphocytes or cancer cells.

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

This method describes how to isolate and immortalize microvascular endothelial cells from mouse brain. We describe a step-by-step protocol starting from the homogenization of brain tissue, digestion steps, seeding and immortalization of the cells. Usually, it takes about five weeks to obtain a homogenous, immortalized microvascular endothelial cell line.

代的永生化小鼠脑微血管内皮细胞株作为<em在体外</em血脑屏障型号

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The ...

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 ...

Olfactory Assays for Mouse Models of Neurodegenerative Disease

In many neurodegenerative diseases and particularly in Parkinson&#8217;s disease, deficits in olfaction are reported to occur early in the disease process and may be a useful behavioral marker for early detection. Earlier detection in neurodegenerative disease is a major goal in the field because this is when neuroprotective therapies have the best potential to be effective. Therefore, in preclinical studies testing novel neuroprotective strategies in rodent models of neurodegenerative disease, olfactory assessment could be highly useful in determining therapeutic potential of compounds and translation to the clinic. In the present study we describe a battery of olfactory assays that are useful in measuring olfactory function in mice. The tests presented in this study were chosen because they measure olfaction abilities in mice related to food odors, social odors, and non-social odors. These tests have proven useful in characterizing novel genetic mouse models of Parkinson&#8217;s disease as well as in testing potential disease-modifying therapies.

Impairment in olfactory function is a common feature in many neurodegenerative disorders including Parkinson, Alzheimer, and Huntington diseases. In the present article, we describe a set of tests for assessing olfaction discrimination and detection in mice that can be used to measure olfactory abilities in mouse models of neurodegenerative diseases.

嗅觉测定的神经退行性疾病的小鼠模型

In many neurodegenerative diseases and particularly in Parkinson&#8217;s disease, deficits in olfaction are reported to occur early in the disease process ...

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene expression in the mouse brain. Here we describe a technique first introduced by Passini and Wolfe for direct intracranial delivery of virally-encoded transgenes into the neonatal mouse brain. In its most basic form, the procedure requires only an ice bucket and a microliter syringe. However, the protocol can also be adapted for use with stereotaxic frames to improve consistency for researchers new to the technique. The method relies on the ability of adeno-associated virus (AAV) to move freely from the cerebral ventricles into the brain parenchyma while the ependymal lining is still immature during the first 12-24 hr after birth. Intraventricular injection of AAV at this age results in widespread transduction of neurons throughout the brain. Expression begins within days of injection and persists for the lifetime of the animal. Viral titer can be adjusted to control the density of transduced neurons, while co-expression of a fluorescent protein provides a vital label of transduced cells. With the rising availability of viral core facilities to provide both off-the-shelf, pre-packaged reagents and custom viral preparation, this approach offers a timely method for manipulating gene expression in the mouse brain that is fast, easy, and far less expensive than traditional germline engineering.

Here we demonstrate a technique for widespread neuronal transduction by intraventricular injection of adeno-associated virus into the neonatal mouse brain. This method provides a rapid and easy way to attain lifelong expression of virally-delivered transgenes.

新生小鼠脑侧脑室注射病毒的持续和广泛的神经元传导

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene ...

Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders

Locomotive defects resulting from neurodegenerative disorders can be a late onset symptom of disease, following years of subclinical degeneration, and thus current therapeutic treatment strategies are not curative. Through the use of whole exome sequencing, an increasing number of genes have been identified to play a role in human locomotion. Despite identifying these genes, it is not known how these genes are crucial to normal locomotive functioning. Therefore, a reliable assay, which utilizes model organisms to elucidate the role of these genes in order to identify novel targets of therapeutic interest, is needed more than ever. We have designed a sensitized version of the negative geotaxis assay that allows for the detection of milder defects earlier and has the ability to evaluate these defects over time. The assay is performed in a glass graduated cylinder, which is sealed with a wax barrier film. By increasing the threshold distance to be climbed to 17.5 cm and increasing the experiment duration to 2 min&#160;we have observed a greater sensitivity in detecting mild mobility dysfunctions. The assay is cost effective and does not require extensive training to obtain highly reproducible results. This makes it an excellent technique for screening candidate drugs in Drosophila mutants with locomotion defects.

We present an optimized inexpensive and reliable negative geotaxis assay in Drosophila melanogaster as a model for neurodegenerative disorders. Being more sensitive to mild locomotor defects, this assay will help screen for potential genetic interactions and drug targets.

攀登缺陷的神经退行性疾病的果蝇模型定量分析

Locomotive defects resulting from neurodegenerative disorders can be a late onset symptom of disease, following years of subclinical degeneration, and ...

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer&#8217;s disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer&#8217;s disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

低聚淀粉样蛋白-β的立体灌注到小鼠海马

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

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 ...

A Human Blood-Brain Interface Model to Study Barrier Crossings by Pathogens or Medicines and Their Interactions with the Brain

The early screening of nervous system medicines on a pertinent and reliable in cellulo BBB model for their penetration and their interaction with the barrier and the brain parenchyma is still an unmet need. To fill this gap, we designed a 2D in cellulo model, the BBB-Minibrain, by combining a polyester porous membrane culture insert human BBB model with a Minibrain formed by a tri-culture of human brain cells (neurons, astrocytes and microglial cells). The BBB-Minibrain allowed us to test the transport of a neuroprotective drug candidate (e.g., Neurovita), through the BBB, to determine the specific targeting of this molecule to neurons and to show that the neuroprotective property of the drug was preserved after the drug had crossed the BBB. We have also demonstrated that BBB-Minibrain constitutes an interesting model to detect the passage of virus particles across the endothelial cells barrier and to monitor the infection of the Minibrain by neuroinvasive virus particles. The BBB-Minibrain is a reliable system, easy to handle for researcher trained in cell culture technology and predictive of the brain cells phenotypes after treatment or insult. The interest of such in cellulo testing would be twofold: introducing derisking steps early in the drug development on the one hand and reducing the use of animal testing on the other hand.

Here we present a protocol describing the setting of an in cellulo BBB (Blood brain barrier)-Minibrain polyester porous membrane culture insert system in order to evaluate the transport of biomolecules or infectious agents across a human BBB and their physiological impact on the neighboring brain cells.

研究病原体或药物交叉障碍及其与大脑相互作用的人血脑界面模型

The early screening of nervous system medicines on a pertinent and reliable in cellulo BBB model for their penetration and their interaction with the ...