这项研究是由德国研究联合会DFG支持下的授权号FO 315/4-1和DFG SFB 688。

1。分离脑微血管<ol><li>对于每一个准备，不论男女，用五至十年的新生小鼠（3-5天）。 </li><li>根据本地IACUC /兽医建议安乐死鼠标，立即取出大脑，并转移到陪替氏培养皿含有下列溶液:15毫摩尔的Hepes（pH值7.4），153 mM氯化钠，5.6 mM KCl中，2.3毫的CaCl 2×2H 2 O，2.6毫摩尔的MgCl 2×6H 2 O（重量/体积），1％牛血清白蛋白（以下称为缓冲液A）。 </li><li>除非另有说明，所有的隔离程序应当在室温（RT）（22-24℃）下在层流罩中进行。一个使用无菌手术刀切的大脑（大脑小脑和脑干无），去除脑膜和毛细管片段，在缓冲。用移液管的组织片段，向上和向下使用10毫升移液管，直到没有团块出现。的悬浮液转移到50毫升Falcon管中并离心，在250×g离心5分钟，在RT。 </li></ol> 注:脑膜可以被确定为薄的，透明的膜的表面处的脑组织。它们可以用无菌镊子小心除去。 <ol start="4"><li>弃上清。 </li><li>将沉淀溶解于4.5ml缓冲液A与1.5毫升的0.75％（w / v的）胶原酶/分散酶（Roche）和孵育45分钟，在37℃下在水浴中（偶尔摇动）。 </li><li>同时，准备通过涂布四个井与IV型胶原（0.1毫克/毫升，溶解在50mM的乙酸）的12孔板。允许坚持1小时。 </li><li>通过加入15毫升冰冷的缓冲液A中重新悬浮的颗粒彻底停止消化。 </li><li>离心该悬浮液在250×g离心10分钟，在RT。 </li><li>弃去上清液。 </li><li>要删除髓鞘，在4°C 1000 xg离心20分钟，加入10毫升25％（W / V）BSA（Sigma公司，纯度&gt; 98％）和离心机25％BSA溶解于1x PBS（PAA实验室）和通过0.2微米f过滤ILTER）。 </li><li>小心弃去上清液，溶解沉淀于10ml缓冲液A，并转移到一个新的Falcon管。 </li><li>离心该悬浮液在250×g离心5分钟，在RT。将所得的颗粒中包含的血管内皮细胞。 </li><li>两倍，Ⅳ型胶原蛋白涂覆的12孔板，用PBS洗净。 </li><li>分为两个井，2毫升至各孔，在4毫升的生长培养基（DMEM中含有10％FCS，50U/ml青霉素/链霉素，1％L-谷氨酰胺）和板的细胞悬浮液中重悬沉淀。的嘌呤霉素添加最终浓度为4微克/毫升，并培育45分钟，在37℃下在细胞培养箱。此步骤允许除去迅速附着细胞。 注:24小时加入嘌呤霉素。只有大脑内皮细胞代谢，为其他类型的细胞，嘌呤霉素是有毒的14。 </li><li>转移2毫升培养基含非粘附细胞的成两个新鲜的井（这些井包含的EC馏分）从步骤1.14。从科技教育的贴壁细胞，填补了井P 1.14与2ml新鲜培养基（这些井包含其他类型的细胞， 如成纤维细胞，星形胶质细胞，可以生长在平行和用于比较的形态）。 </li><li>改变介质的第二天。 </li></ol> 2。名垂千古的脑微血管内皮细胞<ol><li>培养GP + E-86的新（GPENeo）15成纤维细胞，分泌与多瘤病毒中T癌基因的复制缺陷型病毒在DMEM培养基中含有10％热灭活的FCS，50U/ml青霉素/链霉素，和2毫克/毫升的G418（ PAA实验室）在明胶涂瓶中。 注:多瘤中T癌基因转染导致的内皮细胞的生长优势，对非内皮细胞，成均匀的单层细胞与内皮细胞4〜6周的文化形态。 GPENeo不属于市面上可以得到共享的公共材料（请参阅原本发布的4,16）。 </li><li>要使用的含病毒的培养基为<html>永生化，培育GPENeo细胞在G418的无培养基中24小时。 </li><li>取出10毫升的GPEneo上清液并添加聚凝胺（溴化己二甲铵溴化物）（Sigma公司）至最终浓度为8微克/毫升，灭菌通过0.45微米的过滤器，以除去细胞碎片。 </li><li>将生长培养基从准备EC文化的第1部分中，加入2毫升的GPEneo上清液/聚凝胺的混合物入井。第二天重复它的使用新鲜GPEneo上清液/凝聚胺的混合物。 注:聚凝胺是用来使毛孔中的细胞壁，以便在感染的病毒颗粒）。 </li><li>卸下GPEneo上清液/聚凝胺混合物翌日，两次，用PBS洗涤细胞，并维持在生长培养基中的细胞，每3天改变它。汇合后，分裂的细胞1:2Ⅳ型胶原蛋白涂层钢板。 </li><li>通常情况下，稳定脑血管内皮细胞（CEND）线应4-5周后获得。 </li></ol>E"&gt; 3。培养的脑微血管内皮细胞<ol><li>解冻的细胞从cryoaliquot在水浴中，并转移15毫升猎鹰用10ml预先温热的培养基。 </li><li>离心该悬浮液在250×g离心5分钟，在RT。 </li><li>移除介质，传输颗粒中IV型胶原涂覆T 25 cm 2的细胞培养烧瓶与预先温热的生长培养基中（用于电镀的细胞密度至少应为1×10 4细胞/ ml）。 </li><li>改变介质的第二天，达到融合后的细胞分裂细胞（通常在5天）。 </li></ol> 4。 CEND细胞分裂（注:如果一个星期做一次分裂，避免分裂高于1:4）。 <ol><li>取出介质，与PBS洗涤细胞。 </li><li>加入3毫升温暖的胰蛋白酶-EDTA溶液（PAA实验室）（T 75厘米 2烧瓶）中，温育在37℃下，并等待，直到细胞分散层（通常为5至15分钟内）。 </li><li>加入5毫升邻f的生长培养基中，吸取试剂和向下，并转移到一个新的胶原蛋白的IV-涂层的烧瓶中。 </li></ol> 5。冻结的CEND细胞<ol><li>获得的细胞悬浮液，如在步骤4中描述的</li><li>离心该悬浮液在250×g离心5分钟，在RT。 </li><li> 6毫升冷冻培养基中（95％的生长培养基中，5％DMSO）中重悬细胞沉淀（1 T 75 cm 2的烧瓶中，从得到的）。 </li><li>划分到4个1.5毫升冷冻等分试样。 注:可接种于T 25厘米 2烧瓶低温等分的细胞悬液。 </li><li>将冷冻的等分试样储存在液氮下的蒸汽温度。 </li></ol> CEND生长培养基:450毫升DMEM，10％FCS，10毫升L-谷氨酰胺，2％MEM套件，2％NEAA，10毫升钠丙酮酸，50 U / ml青霉素/链霉素 6。代表性的成果的CEND和cerebEND细胞血管内皮一个特点immunostainings届BBB标记以及由跨内皮电阻（TEER）和渗透性的测量。的CEND和cerebEND，脑内皮细胞，原代培养的紧凑细长的细胞的生长抑制汇合单层的形态相似。细胞表达紧密连接蛋白-5，occludin和VE-钙粘蛋白的蛋白质，分别定位在细胞-细胞结以及可检测水平的，由免疫荧光（ 图3）4,5所示。减少血清的培养基中的培养CEND细胞导致增加TEER的（从150欧姆厘米2到第500Ωcm的2存在下，10％血清的存在下，2％血清），这是增强通过加入氢化可的松（800Ωcm的2），或胰岛素（千欧姆厘米2）4。 CEND在减小的血清培养基中培养21天的膜的有TEER的900欧姆厘米2（ 图4）。 TEER使用汇编包含通过电流和电压测量电极（世界精密仪器）。为了进行比较，主微血管脑内皮已报道具有TEER的值的200-600Ωcm的2和市售的小鼠细胞系bEnd.3 TEER的值2（100-140Ωcm的最近的评论见住持2005和Toth 等 2011 人，17,18）。此外，通过降低超过4小时的大分子，如不带电的FITC-葡聚糖的分子量4，10，70和500 kDa的，或荧光素（300沓）跨在2％FCS的分化培养基中的单层CEND相比，与对照细胞保持在含有10％FCS的生长培养基:旁磁通减少对照细胞的30​​％荧光素，至26％，FITC-葡聚糖的10和70 kDa，和磁通被减少到4.5％的对照细胞的FITC标记-dextrtan 500 kDa的。 TEER值相似，渗透率为在GLUC的存在下培养的细胞中的最低水平ocorticoids（GC）4。在进一步的研究中，我们确定了糖皮质激素的靶基因，occludin的，紧密连接蛋白-5和VE-钙粘蛋白在脑血管内皮细胞4,7,9。 GC治疗导致这些蛋白质的表达增加，VE-钙粘蛋白的细胞骨架的重排。直接GC-介导的调控紧密连接蛋白occludin的和claudin-5出现在其启动子区域的4,7,19通过GC反应元件。此外，紧密连接蛋白-5被确定的血管内皮细胞10作为一种新型的雌激素靶。 内皮功能障碍许多不同的疾病的基础。我们氧/葡萄糖剥夺（OGD）条件下培养，CEND。 OGD导致的破坏血脑屏障的功能，这可能会进行重组，综合治疗后与GC和蛋白酶体抑制剂硼替佐米12。 OGD条件导致葡萄糖摄取的强劲增长，货物运输由葡萄糖的表达TERS在cerebEND，这可能是由此外MK801，非竞争性NMDA-受体抑制剂13衰减。 <img alt="图1" src="/files/ftp_upload/4022/4022fig1.jpg" /> 图1。 （A）的6倍和15倍（B）的放大倍率下脑微血管内皮细胞（CEND）隔离一个星期后。光镜图像的形态 。岛上血管内皮细胞形成的菌落是可见的。 <img alt="图2" src="/files/ftp_upload/4022/4022fig2.jpg" /> 图2。形态的脑微血管内皮细胞（CEND）1个月后，隔离和永生。光镜图片放大15倍。可以观察到一个融合的，均匀的内皮细胞单层。 <img alt="图3" src="/files/ftp_upload/4022/4022fig3.jpg" /> 图3。永生大脑microvasculAR内皮细胞表达紧密连接蛋白-5，occludin和VE-cadherin的表达。Ⅳ型胶原涂层盖玻片上CEND细胞生长和抗体的紧密连接蛋白-5（A）（B），紧密连接蛋白和VE-钙粘蛋白（C）染色。影像拍摄使用蔡司Axioscop2的40倍放大的显微镜下。 <img alt="图4" src="/files/ftp_upload/4022/4022fig4.jpg" /> 图4。 Ⅳ型胶原涂层Transwell小的过滤器（孔径为0.4微米） 测量跨内皮电阻（TEER）CEND单层。CEND的的生长。达到汇合后，将细胞保持在含2％FCS的培养基中。 TEER测量后，7日，14日和21日，使用一个程序集包含通过电流和电压测量电极。

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Cancer Res. 64, 125 (1994).</li><li>Kiefer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cell+transformation+by+polyomavirus+middle+T+antigen+in+mice+lacking+Src-related+kinases.">Endothelial cell transformation by polyomavirus middle T antigen in mice lacking Src-related kinases.</a> Curr. Biol. 4, 100 (1994).</li><li>Ong, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ShcA+and+Grb2+mediate+polyoma+middle+T+antigen-induced+endothelial+transformation+and+Gab1+tyrosine+phosphorylation.">ShcA and Grb2 mediate polyoma middle T antigen-induced endothelial transformation and Gab1 tyrosine phosphorylation.</a> EMBO J. 20, 6327 (2001).</li><li>O'Connell, K. A., Edidin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+lymphoid+endothelial+cell+line+immortalized+by+simian+virus+40+binds+lymphocytes+and+retains+functional+characteristics+of+normal+endothelial+cells.">A mouse lymphoid endothelial cell line immortalized by simian virus 40 binds lymphocytes and retains functional characteristics of normal endothelial cells.</a> J. Immunol. 144, 521 (1990).</li><li>Roux, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+gamma-glutamyl+transpeptidase+and+alkaline+phosphatase+activities+in+immortalized+rat+brain+microvessel+endothelial+cells.">Regulation of gamma-glutamyl transpeptidase and alkaline phosphatase activities in immortalized rat brain microvessel endothelial cells.</a> J. Cell Physiol. 159, 101 (1994).</li><li>Montesano, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+proteolytic+activity+is+responsible+for+the+aberrant+morphogenetic+behavior+of+endothelial+cells+expressing+the+middle+T+oncogene.">Increased proteolytic activity is responsible for the aberrant morphogenetic behavior of endothelial cells expressing the middle T oncogene.</a> Cell. 62, 435 (1990).</li><li>Steiner, O., Coisne, C., Engelhardt, B., Lyck, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+immortalized+bEnd5+and+primary+mouse+brain+microvascular+endothelial+cells+as+in+vitro+blood-brain+barrier+models+for+the+study+of+T+cell+extravasation.">Comparison of immortalized bEnd5 and primary mouse brain microvascular endothelial cells as in vitro blood-brain barrier models for the study of T cell extravasation.</a> J. Cereb. Blood Flow Metab. 31, 315 (2010).</li><li>Skaper, S. D., Conn, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Neurosciences. 2, 1303 (1990).</li><li>Ishii, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thrombomodulin,+an+endothelial+anticoagulant+protein,+is+absent+from+the+human+brain.">Thrombomodulin, an endothelial anticoagulant protein, is absent from the human brain.</a> Blood. 67, 362 (1986).</li><li>Minagar, A., Alexander, J. S. <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+disruption+in+multiple+sclerosis.">Blood-brain barrier disruption in multiple sclerosis.</a> Mult. Scler. 9, 540 (2003).</li></ol>

没有利益冲突的声明。

所描述的程序可用于隔离的微血管内皮细胞从不同的小鼠品系，以及从不同的基因敲除小鼠品系，研究具体的血管内皮功能的变化。 作为永生的方法，我们采用了与癌基因的小鼠多瘤病毒，多瘤中T抗原转变。迅速转化未成熟的血管内皮细胞， 在体内和体外 20,21,22。在文献中描述的内皮细胞的永生化的其它方法包括，例如，丝棉空泡病毒SV40大T抗原40 23，腺病毒基因产物E1A 24或过度的催化亚基端粒酶3的永生化。的永生与PymT是特异于小鼠未成熟的内皮细胞，这可以从新生小鼠在很短的时间内获得均匀的EC文化。永生化的改变的细胞的性质和th与原代细胞或小鼠体内的实验中得到永生细胞株的E结果应该是比较。 PymT永生已被形容为EC表达高水平的纤维蛋白溶解活性，导致尿激酶型纤溶酶原激活物增加生产，降低生产型纤溶酶原激活物抑制剂25。直接比较PymT永生bEND5与初级内皮细胞系表明血脑屏障体外模型非常适用于T细胞的粘附研究26。 建立的细胞系，根据所描述的过程，可以使用在低通道数保持阻隔性能通过BBB和EC交界蛋白质的高表达的，例如细胞衰老过程中失去了这些属性。因此阻隔性能的损失后，应考虑编制一个新的细胞系。应该是高内皮细胞接种密度，因为这是细胞增殖的关键。在分离过程中，必须考虑以及维护的永生化内皮细胞。应测试每一个新的细胞系，其阻隔性和与其他类型的细胞的可能的污染物。 EC单层可与作为第一抗体的抗-半乳糖脑苷，抗神经胶质原纤维酸性蛋白和抗平滑肌抗原染色排除污染的少突胶质细胞，星形胶质细胞和周细胞4,27。为了排除污染meningal脉管，血栓调节蛋白的表达，可以进行测试，这是表示在所有血管床的与异常的脑28。 有趣的是，永生微血管内皮细胞系中分离出不同的大脑区域不同，其阻隔性和敏感性，有利于炎症的刺激。脑CEND和小脑cerebEND的细胞系作为一个例子，可以举出4,5。 CerebEND表明，在比较CEND，主要紧密连接的部件紧密连接蛋白1和occludin的表达水平较低。不过，该水平的claudin-3和-12在cerebEND高。的屏障功能cerebEND细胞遭受更与炎症介体的处理下，TNFα比的阻隔性能CEND细胞做了5。高等小脑炎症刺激的脆弱性，可以观察到在体内在多发性硬化症患者和动物模型的实验性自身免疫性脑脊髓炎29。这些有趣的发现表明，在为了改善未来的靶向药物进入大脑产生和表征个人的体外模型不同脑区的必要性。

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>牛血清白蛋白（BSA） </td><td> Sigma-Aldrich公司</td><td> A7906 </td><td>纯度&gt; 98％ </td></tr><tr><td> Ⅳ型胶原</td><td> Sigma-Aldrich公司</td><td> C5533 </td><td>在50mM的乙酸的50微克/毫升</td></tr><tr><td>胶原酶/ Dispase酶蛋白酶</td><td>罗氏公司</td><td> 10269638001 </td><td></td></tr><tr><td> Dulbecco改良的Eagle培养基（DMEM） </td><td> Sigma-Aldrich公司</td><td> D5796 </td><td></td></tr><tr><td>二甲亚砜（DMSO） </td><td> Sigma-Aldrich公司</td><td> D2650 </td><td></td></tr><tr><td>小牛血清（FCS） </td><td> PAA实验室</td><td> A15110-1333 </td><td>终浓度10％，热失活（30分钟，在56℃下） </td></tR&gt; <tr><td> L-谷氨酰胺</td><td> Biochrom AG </td><td> K0282 </td><td>存储:≤-15°C </td></tr><tr><td> MEM维生素</td><td> Biochrom AG </td><td> K0373 </td><td>存储:≤-15°C </td></tr><tr><td>丙酮酸钠</td><td> Biochrom AG </td><td> L0473 </td><td></td></tr><tr><td>新霉素（G418） </td><td> PAA实验室</td><td> P11-012 </td><td></td></tr><tr><td>非必需氨基酸（NEA） </td><td> Biochrom AG </td><td> K0293 </td><td>储存在4°C </td></tr><tr><td>青霉素/链霉素</td><td> Biochrom AG </td><td> A2212 </td><td>存储:≤-15°C </td></tr><tr><td>磷酸盐缓冲盐水（PBS） </td><td> Biochrom AG </td><td> L1825 </td><td></td></tr><tr><td>聚凝胺（溴化己二甲铵溴） </td><td> Sigma-Aldrich公司</td><td> 107689 </td><td> 0.8毫克/毫升在PBS中，在-20℃下贮存</td></tr><tr><td>嘌呤</td><td> Sigma-Aldrich公司</td><td> P8833 </td><td></td></tr><tr><td>胰蛋白酶/ EDTA </td><td> Biochrom AG </td><td> L1825 </td><td> 0.05％，0.02％EDTA在PBS中</td></tr></tbody></table>

generation of an immortalized murine brain microvascular endothelial cell line as an in vitro blood brain barrier model

上皮细胞和内皮细胞（EC）的建筑旁的壁垒保护组织的外部和内部环境。血脑屏障（BBB）的EC，星形胶质细胞最终脚，周细胞和基底膜负责保护和脑实质的动态平衡。， 在体外 BBB车型的常用工具BBB的结构和功能的研究在细胞水平上。在不同的实验室的研究有相当多的不同的体外 BBB模型已经建立。一般，将细胞从牛，猪，大鼠或小鼠的脑组织（Wilhelm 等中详细讨论的评论1）获得的。只适用于有限数量的实验室或公司2,3人体组织样本。虽然原代细胞的准备工作耗时和EC文化的不同的批次，建立永生EC林ES的重点是对科学的兴趣。 在这里，我们提出了建立一个永生的脑微血管EC线从新生鼠脑的方法。我们描述的步骤一步一步上市的试剂和解决方案。由我们的实验室确定的方法允许均匀的永生化的四到五个星期内的内皮细胞株的隔离。脑微血管内皮细胞株被称为CEND 4（大脑皮层）和cerebEND 5（从小脑皮质），根据此过程中福斯特实验室分离并已有效地用于解释不同的生理和病理过程中的BBB。使用CEND和cerebEND我们已经证明，这些细胞对糖皮质激素4,6-9和雌激素治疗10到的亲infammatory介质，如TNFalpha 5,8。此外，我们还研究了病理多个SClerosis的EC-11和缺氧12,13。的的CEND和cerebEND线可以被认为是一个很好的工具为不同的刺激，互动的EC细胞或肿瘤细胞的结构和功能研究的BBB内皮细胞，细胞的反应。

Watch this Scientific Journal Video about Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model at JoVE.com

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

此方法描述了如何从小鼠脑微血管内皮细胞隔离和不朽。我们描述了一个一步一步的协议，从同质化的脑组织，消化步骤，播种和永生化的细胞。通常情况下，大约需要五周获得一个同质的，永生的微血管内皮细胞系。

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 ...

generation-an-immortalized-murine-brain-microvascular-endothelial

Research

JoVE Journal

Neuroscience

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

18.8K 次观看.  University of Wurzburg. 此方法描述了如何从小鼠脑微血管内皮细胞隔离和不朽。我们描述了一个一步一步的协议，从同质化的脑组织，消化步骤，播种和永生化的细胞。通常情况下，大约需要五周获得一个同质的，永生的微血管内皮细胞系。

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

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

Design and Assembly of an Ultra-light Motorized Microdrive for Chronic Neural Recordings in Small Animals

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of neural circuits underlying a variety of natural behaviors, including navigation1 , decision making 2,3, and the generation of complex motor sequences4,5,6. Advances in precision machining has allowed for the fabrication of light-weight devices suitable for chronic recordings in small animals, such as mice and songbirds. The ability to adjust the electrode position with small remotely controlled motors has further increased the recording yield in various behavioral contexts by reducing animal handling.6,7
 Here we describe a protocol to build an ultra-light motorized microdrive for long-term chronic recordings in small animals. Our design evolved from an earlier published version7, and has been adapted for ease-of use and cost-effectiveness to be more practical and accessible to a wide array of researchers. This proven design 8,9,10,11 allows for fine, remote positioning of electrodes over a range of ~ 5 mm and weighs less than 750 mg when fully assembled. We present the complete protocol for how to build and assemble these drives, including 3D CAD drawings for all custom microdrive components.

The design, fabrication and assembly of an ultra-light motorized microdrive is described. The device provides a cost-effective and easy-to-use solution for chronic recordings of single units in small behaving animals.

设计和组装的超轻型电动微型硬盘（Microdrive）慢性神经录音的小动物

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of ...

Isolation of Primary Murine Brain Microvascular Endothelial Cells

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional complex of tight and adherens junctions. Together with other cell-types such as astrocytes or pericytes, they form the neurovascular unit (NVU), which specifically regulates the interchange of fluids, molecules and cells between the peripheral blood and the CNS. Through this complex and dynamic system BMECs are involved in various processes maintaining the homeostasis of the CNS. A dysfunction of the BBB is observed as an essential step in the pathogenesis of many severe CNS diseases. However, specific and targeted therapies are very limited, as the underlying mechanisms are still far from being understood. 
Animal and in vitro models have been extensively used to gain in-depth understanding of complex physiological and pathophysiological processes. By reduction and simplification it is possible to focus the investigation on the subject of interest and to exclude a variety of confounding factors. However, comparability and transferability are also reduced in model systems, which have to be taken into account for evaluation. The most common animal models are based on mice, among other reasons, mainly due to the constantly increasing possibilities of methodology. In vitro studies of isolated murine BMECs might enable an in-depth analysis of their properties and of the blood-brain-barrier under physiological and pathophysiological conditions. Further insights into the complex mechanisms at the BBB potentially provide the basis for new therapeutic strategies.
This protocol describes a method to isolate primary murine microvascular endothelial cells by a sequence of physical and chemical purification steps. Special considerations for purity and cultivation of MBMECs as well as quality control, potential applications and limitations are discussed.

Brain microvascular endothelial cells (BMEC) are interconnected by specific junctional proteins forming a highly regulated barrier separating blood and the central nervous system (CNS), the so-called blood-brain-barrier (BBB). The isolation of primary murine brain microvascular endothelial cells, as discussed in this protocol, enables detailed in vitro studies of the BBB.

原代鼠脑微血管内皮细胞的分离

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional ...

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the gold-standard experimental model to study basic neurophysiological mechanisms involved in the formation and maintenance of neuronal cell assemblies. However, in vitro studies have been limited to the recording of the electrophysiological activity generated by bi-dimensional (2D) neural networks. Nonetheless, given the intricate relationship between structure and dynamics, a significant improvement is necessary to investigate the formation and the developing dynamics of three-dimensional (3D) networks. In this work, a novel experimental platform in which 3D hippocampal or cortical networks are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are realized by seeding neurons in a scaffold constituted of glass microbeads (30-40 &#181;m in diameter) on which neurons are able to grow and form complex interconnected 3D assemblies. In this way, it is possible to design engineered 3D networks made up of 5-8 layers with an expected final cell density. The increasing complexity in the morphological organization of the 3D assembly induces an enhancement of the electrophysiological patterns displayed by this type of networks. Compared with the standard 2D networks, where highly stereotyped bursting activity emerges, the 3D structure alters the bursting activity in terms of duration and frequency, as well as it allows observation of more random spiking activity. In this sense, the developed 3D model more closely resembles in vivo neural networks.

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

In this work, a novel experimental model in which 3D neuronal cultures are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are built by seeding neurons in a scaffold made up of glass microbeads on which neurons grow and form interconnected 3D structures.

接口3D工程神经细胞微电极阵列:一种创新<em&gt;体外</em&gt;实验模型

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the ...

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol demonstrates the implementation of an optimized N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. A single media formulation is used to reliably obtain healthy brain slices from animals of any age and for diverse experimental applications.

用优化的n-甲基 d-葡乙胺保护恢复法制备急性脑切片

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...

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 ...

Isolation of Primary Murine Skeletal Muscle Microvascular Endothelial Cells

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal muscles regulating the exchange of fluids and nutrients as well as the immune response against infectious agents by controlling immune cell migration. For these functions, MMEC form a functional &#34;myovascular unit&#34; (MVU), with further cell types, such as fibroblasts, pericytes and skeletal muscle cells. Consequently, a dysfunction of MMEC and therefore the MVU contributes to a vast variety of myopathies. However, regulatory mechanisms of MMEC in health and disease remain insufficiently understood and their elucidation precedes more specific treatments for myopathies. The isolation and in-depth investigation of primary MMEC functions in the context of the MVU might facilitate a better understanding of these processes.
This article provides a protocol to isolate primary murine MMEC of the skeletal muscle by mechanical and enzymatic dissociation including purification and culture maintenance steps.

Microvascular endothelial cells of skeletal muscles (MMEC) shape the inner wall of muscle capillaries and regulate both, exchange of fluids/molecules and migration of (immune) cells between muscle tissue and blood. Isolation of primary murine MMEC, as described here, enables comprehensive in vitro investigations of the &#34;myovascular unit&#34;.

原代小鼠骨骼肌微血管内皮细胞的分离

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal ...

A Preclinical Model to Assess Brain Recovery After Acute Stroke in Rats

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery occlusion/reperfusion (MCAO/R) model in male Sprague-Dawley rats was chosen. By changing the rat&#39;s weight (260&#8722;330 g), the thread bolt type (2636/2838/3040/3043) and the brain infarct time (2-3 h), a higher Longa&#39;s score, a larger infarct volume and a greater model success ratio were screened using the Longa&#39;s score and TTC staining. The optimum model condition (300 g, 3040 thread bolt, 3 h brain infarct time) was acquired and used in a 1-90 day observation period after reperfusion via assessment of sensorimotor functions and infarct volume. At these conditions, the bilateral asymmetry test had a significant difference from 1 to 90 days, and the grid-walking test had a significant difference from 1 to 60 days; both differences could be a suitable sensorimotor functional test. Thus, the most appropriate condition of a novel rat model in the recovery and sequela stages of brain ischemia was found: 300 g rats that underwent MCAO with a 3040 thread bolt for a 3 h brain infarct and then reperfused. The appropriate sensorimotor functional tests were a bilateral asymmetry test and a grid-walking test.

The&#160;purpose of this study is to establish and validate an animal model for research in the recovery and sequela stages of brain ischemia by testing brain infarction and sensorimotor function after middle cerebral artery occlusion/reperfusion (MCAO/R) after 1-90 days in rats.

评估大鼠急性中风后脑恢复的临床前模型

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery ...

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.