NINDS补助NS49720和NS65819和NCI授予CA97257

1。肿瘤细胞的制备。 源自人类大脑肿瘤异种移植的细胞可以从肿瘤在裸鼠皮下生长，或从细胞培养繁殖。在下面讨论这两种细胞来源的利用，随着细胞移植的方法示范。 为了准备皮下肿瘤细胞转移到颅内车厢，切除侧翼肿瘤置于培养皿中，组织最初是用手术刀，然后重复吹打创建一个细胞总悬浮 1机械打乱剁碎。细胞总悬浮液，然后通过一个70μm的尼龙网过滤，生产单细胞悬液，适合颅内注射。细胞悬液，离心1000转10分钟，在4 ° C，并resuspending适当体积的无血清培养基中的细胞沉淀，获得了最后的工作浓度（见下文）前吸气上清。对于准备建立的细胞系颅内植入，细胞的收获trypsinizing单层，或通过收集神经球悬浮培养，然后离心和resuspending 以上2所示的细胞。注射细胞的数量是注射神经解剖学位置上的变量的依赖。幕注射，我们经常注入3μL无血清培养基（DMEM）3-5 × 10 5细胞，而脑干注射 3，如5 × 10 4细胞数在0.5μL注入。注射比建议大量可导致肿瘤细胞通过针道返流，由此exophytic（图1），而不是颅内肿瘤的生长。撤销颅内注射样品后，剩余的细胞悬液，应放在冰，混合，经常保持适当的浓度，同时完成注塑系列成员之间的颅内肿瘤建立的内容。 2。肿瘤细胞移植请注意，下面描述的所有程序已经审查和批准的机构使用动物，并在加州大学旧金山分校护理委员会。 <ol><li>手术区应做好所有的表面，如2％洗必泰溶液，用消毒剂喷洒。使用消毒剂后，应放在以下耗材在手术区： <ul><li>加热垫，以保持鼠标体温</li><li>两个小培养皿;一个含有3％的双氧水，和一个含有2％洗必泰</li><li>无菌纱布和棉签</li><li>无菌一次性手术刀</li><li>蒸压手术吻合器</li></ul></li><li>麻醉应采用注射麻醉剂，通常是氯胺酮，甲苯噻嗪的混合物。 </li><li>一旦鼠标被麻醉，头皮是准备用一块无菌纱布蘸洗必泰溶液擦拭几次。眼药膏应在手术过程中保持充足的水分。使用无菌手术刀，完成了parieto枕骨一个矢状切口，约1厘米长。暴露颅骨表面，然后用棉签在3％的双氧水溶液浸泡清洗。前囟门应在这一点上明显（见视频）。 </li><li>肿瘤细胞注射的坐标会有所不同，根据肿瘤的建立所需的站点。下面我们使用脑肿瘤建立在神经解剖学位置，在许多脑肿瘤患者的经验肿瘤的发生发展的过程。在脑肿瘤研究感兴趣的其他地点包括脑桥，脑干肿瘤 3解剖模型，并硬膜下注射建模脑膜肿瘤的位置。肿瘤细胞注射前，用无菌的25计利针穿刺的头骨在2 mm至前囟门，冠状缝前1毫米的权利，从而创造一个注射肿瘤细胞（见视频）开放。此过程可以很好地用于小鼠和大鼠的老鼠（22号针头）。 </li><li>进入注射器吸取细胞之前，用手指轻敲混合细胞悬液的内容。与所需数量的细胞悬液装入注射器，小心，以避免产生气泡。注射器外，然后用酒精棉球清洗擦拭外观无任何贴壁细胞，这将有助于防止颅外肿瘤的建立和成长（图1A）。为了确保适当的注射深度取得，使用手术刀切割3mm的关闭了P20的pipetteman提示指出。本节针尖可以装在注射器，将采取措施限制注射深度，此外，并会确保该TI注射器针头P是从3mm的头骨底部。注射器垂直放置的头骨，并在以前创建的孔，慢慢注入细胞悬液（超过1分钟的时间内注入一个3μL应暂停）。注射器插入一个不恰当的角度，可能会导致在完成注射后的细胞和随后脊髓传播（图1B：右）脑室注射，针留在一分钟的地方，然后慢慢退出（见视频）：这些步骤将有助于减少肿瘤细胞的回流。 </li><li>作为替代求告无门或手绘的方法颅内肿瘤细胞植入 5，可以使用小动物脑立体定向仪（面板的示意图F）的，一般促进更一致的注射位置，但在大量的费用程序的时间。在我们的经验，两个手术人员可以注入多达60只小鼠/小时，在使用自由的做法，而用小动物脑立体定向仪的最大注射率约为15只/小时。程序权宜之计，是一个重要的考虑因素时，小鼠注射大系列，并有助于减少肿瘤细胞，被注入，在冰上剩余的时间。 </li><li> 3％双氧水和使用无菌干棉签干燥清洁的头骨。应用无菌骨蜡孔。使用镊子，共同绘制头皮以上的头骨和主食关闭。为了达到最佳的愈合，头皮应装订，每一方对对方的头皮的真皮层（对底面底面）。装订头皮应清理干净，用洗必泰溶液，然后通过手术后的疼痛缓解皮下注射管理和丁丙诺啡。 </li><li>手术后，直到他们成为园内，并保留正常活动监视所有小鼠。通常情况下，恢复时间约为30分钟。 </li></ol> 3。生物发光监测肿瘤生长<ol><li>背景。生物发光成像（劳保局）是基于荧光素的氧化[D -（ - ）-2 - （60 - 羟基- 20 -苯并噻唑） - 噻酮-4 - 羧酸的氧气和三磷酸腺苷（ATP）的存在。这是催化反应的酶的荧光素酶，转换成化学能，由此产生的排放光的光子。人体细胞表达荧光素酶与荧光素酶表达的细胞能够发光的荧光素底物的存在（见下文），可以修改。是从动物的最小的背景发光的主机荧光素治疗，例如，有一个非常有利的信号噪声比检测荧光素酶修饰的肿瘤细胞发光的排放量，使高度敏感的肿瘤的生长和体内治疗监测的。此外，荧光素酶及其底物，荧光素，已被证明非哺乳动物细胞的毒性，和我们所观察到相应的未修改的细胞表达荧光素酶相对的细胞之间功能上没有区别。荧光素很容易穿过细胞膜和后腹腔内的血 - 脑屏障（IP）或静脉（IV）注射液在小鼠体内，从而使每一个车厢，其中包含荧光素酶基因细胞成像。光子发射和荧光素酶基因细胞所发出的光频谱的水平是足够的穿透组织的研究小动物，如小鼠和大鼠，，并与低光成像摄像机可以检测外部。生物发光成像的非侵入性的性质，允许反复监测个别受试动物治疗肿瘤的生长和响应。 </li><li>萤火虫荧光素酶的表达，植入的细胞来源应该是稳定的修改。可以购买这种细胞来源，或个人利用已构建荧光素酶的组成型表达的慢病毒实验室。我们强烈建议使用的荧光素酶编码的慢病毒，而不是质粒，修饰细胞，以确保稳定的荧光素酶的细胞在体内的表达，这是必要的定量发光成像在肿瘤负荷提供准确地反映动物个体的课题，反复的变化在实验过程成像。 </li><li>劳保局监测。我们建议进行定量生物发光成像（qBLI）1X - 2X每周，肿瘤细胞注射后的一个星期开始。我们qBLI进行使用IVIS Lumina的成像站（卡尺生命科学版），和我们的研究结果表明，类似的数据是在使用IVIS Lumina的，IVIS 150，或IVIS 200成像站。在准备用于成像，老鼠同时通过腹腔注射麻醉与氯胺酮/甲苯​​噻嗪和管理的荧光素（D -荧光素盐，钾150毫克/公斤，卡尺生命科学），成像在注射后12分钟的小鼠。在荧光素的药代动力学表明，adminis之间的时间细胞发光的荧光素和决心tration应在10-15分钟之间的荧光素注射后，以获得最大的发光排放和最大的检测灵敏度。 10-15分钟的时间间隔内选定的时间长度应保持常数正在成像动物之间。这是非常重要的是保持在注射荧光素和获得BLI读数之间的时间长度的一致性。包括颅内信号区域利益的定义区域使用的生活图像软件（图2），总photons/s/sr/cm2（每平方厘米每秒每球面度的光子）的记录（见视频）。 </li><li>数据分析。个别动物进行监测，而肿瘤的生长和治疗的反应，我们强烈建议增加肿瘤反应的统计结论的确定性至少有8个治疗组，或缺乏，治疗。方面介绍qBLI结果，发光读数转换为标准化值除以每个鼠标小号发光，期间获得的治疗方案完成后，其相应的最大预处理读6,7发光。生存分析，采用Kaplan - Meier估计8使用，并从中产生生存曲线，中位生存期值确定。使用log - rank检验比较生存曲线之间的差异。 </li><li>随后的分析处理和未经处理的肿瘤脑检索。动物安乐死后，应迅速切除鼠脑（见视频），并在福尔马林放在肿瘤的形态学和免疫组织化学的后续分析，或应安装在华侨城标本冻结。 </li></ol> 4。代表性的成果如图3a所示的例子中，接受颅内肿瘤细胞注射的小鼠进行了监测颅内发光，直到连续平均发光值表示进步的肿瘤生长，此时治疗开始（第34天开始的灰色箭头：厄洛替尼每日150毫克管理直到要求安乐死/公斤）。每个鼠标的发光值设置为1的归一化值，在开始治疗时，每个鼠标以后归其最终的预处理成像价值发光读数。作为一个例子，鼠标与治疗前的2.0 × 10 7光子/秒34天，其发光已上升至6.0最后的发光读数× 10 7光子/天38秒，每天38归发光值3.0。平均正常化生物发光和相应的控制和治疗组的标准误差为每个成像时间点绘制。在这个例子中，均归发光的显着性差异明显，在第一成像时间点后开始治疗（38天），平均组在随后的时间点进一步提高发光的区别。在大多数情况下，抗肿瘤治疗，qBLI表示，活动是伴随着相应的生存显着性差异（即P &lt;0.05），是这里的情况（图3B）。面板3C和3D显示相邻的苏木精和曙红和鼠脑的抗EGFR染色切片获得的“安乐死”时，福尔马林和随后的石蜡包埋后切片手术切除脑安置。 <img src="/files/ftp_upload/1986/1986fig1.jpg" alt="图1" /> 图1。颅内注射错误的迹象。一）Exophytic（颅外）肿瘤的生长（红色圆圈），可以造成太大的注射量，剩余的细胞悬液注射器，或从注射器注射肿瘤细胞后，撤回太快。二）注入脑室肿瘤细胞，可导致脊柱肿瘤的传播（鼠标右），相反，正确注射的肿瘤细胞，为保持局部注射部位（鼠标左）信号。 <img src="/files/ftp_upload/1986/1986fig2.jpg" alt="图2" /> 图2（右）代表从控制老鼠的生物发光强度交涉热图图像（左）和治疗组治疗的反应实验。生活图像软件可以设置定义感兴趣区域（红色圆圈），或仪器操作员可以手动定义感兴趣区域。对于使用如施工图这些图像，我们建议，仪器操作员显示热图的图像，使用相同的生物发光的热图范围（图上半部分），提供的可视化表示的生物发光受试动物之间的差异程度。 <img src="/files/ftp_upload/1986/1986fig3.jpg" alT =“图3”/&gt; 图3。生物发光，生存和肿瘤组织分析，从实验中，治疗反应是显而易见的。剧情的控制和治疗组小鼠的平均生物发光读数与标准错误，的），表示每个成像点。 B）相同的小鼠的生存情节; P -值确定通过使用log - rank 检验 7 。 C）H＆E染色部分的鼠脑肿瘤。四）EGFR染色部分。 E和F）表示地区倍率板彗星和D，分别与面板显示肿瘤抑制蛋白PTEN的负染发送。

<ol>
	<li>Giannini, C., Sarkaria, J. N., Saito, A., Uhm, J. H., Galanis, E., Carlson, B. L., Schroeder, M. A., James, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient+Tumor+EGFR+and+PDGFRA+Gene+Amplifications+Retained+in+an+Invasive+Intracranial+Xenograft+Model+of+GBM.">Patient Tumor EGFR and PDGFRA Gene Amplifications Retained in an Invasive Intracranial Xenograft Model of GBM.</a> Neuro-Oncol. 7, 164-176 (2005).</li><li>Ozawa, T., Wang, J., Hu, L. J., Bollen, A. W., Lamborn, K. R., Deen, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+human+glioblastomas+as+xenografts+in+the+brains+of+athymic+rats.">Growth of human glioblastomas as xenografts in the brains of athymic rats.</a> In Vivo. 1, 55-60 (2002).</li><li>Hashizume, R., Ozawa, T., Dinca, E. B., Banerjee, A., Prados, M. D., James, C. D., Gupta, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+human+brainstem+glioma+xenograft+model+enabled+for+bioluminescence+imaging.">A human brainstem glioma xenograft model enabled for bioluminescence imaging.</a> J Neurooncol. , (2009).</li><li>Baia, G. S., Dinca, E. B., Ozawa, T., Kimura, E. T., McDermott, M. W., James, C. D., VandenBerg, S. R., Lal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+orthotopic+skull+base+model+of+malignant+meningioma.">An orthotopic skull base model of malignant meningioma.</a> Brain Pathol. 2, 172-179 (2007).</li><li>Ozawa, T., James, C. D., Van Meir, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Brain+Tumor+Cell+and+Tumor+Tissue+Transplantation+Models.">Human Brain Tumor Cell and Tumor Tissue Transplantation Models.</a> CNS Cancer: Models, Markers, Prognostic Factors, Targets, and Therapeutic Approaches. , 147-162 (2009).</li><li>Sarkaria, J. N., Yang, L., Grogan, P. T., Kitange, G. J., Carlson, B. L., Schroeder, M. A., Galanis, E., Giannini, C., Wu, W., Dinca, E. B., James, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Molecular+Characteristics+Correlated+with+Glioblastoma+Sensitivity+to+EGFR+Kinase+Inhibition+Through+Use+of+an+Intracranial+Xenograft+Test+Panel.">Identification of Molecular Characteristics Correlated with Glioblastoma Sensitivity to EGFR Kinase Inhibition Through Use of an Intracranial Xenograft Test Panel.</a> Mol. Cancer Ther. 6, 1167-1174 (2007).</li><li>Dinca, E. B., Sarkaria, J. N., Schroeder, M. A., Carlson, B. L., Voicu, R., Gupta, N., Berger, M. S., James, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence+Monitoring+of+Intracranial+Glioblastoma+Xenograft+Response+to+Primary+and+Salvage+Temozolomide+Therapy.">Bioluminescence Monitoring of Intracranial Glioblastoma Xenograft Response to Primary and Salvage Temozolomide Therapy.</a> J. Neurosurg. 107, 610-616 (2007).</li><li>Kaplan, E. L., Meier, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-parametric+estimation+from+incomplete+observations.">Non-parametric estimation from incomplete observations.</a> J. Am. Stat. Assoc. 53, 457-481 (1958).</li><li>Peto, R., Peto, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asymptotically+efficient+rank+invariant+procedures.">Asymptotically efficient rank invariant procedures.</a> J. R. Stat. Soc. Ser. A. Stat. Soc. 135, 185-207 (1972).</li><li>Camphausen, K., Purow, B., Sproull, M., Scott, T., Ozawa, T., Deen, D. F., Tofilon, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+in+vivo+growth+on+human+glioma+cell+line+gene+expression:+convergent+profiles+under+orthotopic+conditions.">Influence of in vivo growth on human glioma cell line gene expression: convergent profiles under orthotopic conditions.</a> Proc. Natl. Acad. Sci. U. S. A.. 102, 8287-8292 (2005).</li></ol>

原位（颅内）脑肿瘤异种移植的建立提供了一个适当的微环境建模中枢神经系统癌症治疗反应测试 10 。这种类型的造型，另外还提供关于治疗获得脑和脑肿瘤，这是非常重要的，以确定是否应提前在患者的临床试验评价实验代理的信息。由于颅内移植瘤的金额不能直接测量，如卡尺，纵向监测的颅内肿瘤的生长和对治疗的反应要求与我们的经验表明生物发光成像的非侵入性的成像，最实用的方法进行实验，其主要目标是评估肿瘤对治疗的反应程度。当生物发光成像的结果与动物受生存分析相结合，这两个数据集提供了一个强大和可靠的实验疗效评估的方法。 最后，这是极为重要的颅内脑肿瘤异种移植，安乐死受试动物的收获，以评估治疗的形态学和分子的影响，为此，我们希望在实施安乐死时全脑切除术，切除大脑保存以供后续分析，。 鉴于前面的介绍已经取得了具体的脑瘤研究，概念肯定generalizeable适合在啮齿类动物中的原位模型​​的其他人类癌症。

<table border="1">
	<thead>
		<tr>
			<th>Material Name</th>
			<th>Type</th>
			<th>Company</th>
			<th>Catalogue Number</th>
			<th>Comment</th>
		</tr>
	</thead>
	<tbody>
		<tr>
			<td>Disposable Scapels</td>
			<td>&nbsp;</td>
			<td>Feather</td>
			<td>2975</td>
			<td>No.21</td>
		</tr>
		<tr>
			<td>Heating Pad</td>
			<td>&nbsp;</td>
			<td>Dunlap</td>
			<td>HP950</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Gauze</td>
			<td>&nbsp;</td>
			<td>Fisher Scientific</td>
			<td>22028563</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cotton Swabs</td>
			<td>&nbsp;</td>
			<td>Fisher Scientific</td>
			<td>23-400-100</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>2% Chlorhexidine</td>
			<td>&nbsp;</td>
			<td>Fisher Scientific</td>
			<td>NC9756995</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>3% Hydrogen Peroxide</td>
			<td>&nbsp;</td>
			<td>Fisher Scientific</td>
			<td>H312P-4</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>25g Needle</td>
			<td>&nbsp;</td>
			<td>Becton Dickinson</td>
			<td>305122</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>10ul Hamilton Sharp Microsyringe</td>
			<td>&nbsp;</td>
			<td>Hamilton</td>
			<td>20734</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Skin Stapler and Staples</td>
			<td>&nbsp;</td>
			<td>Stoelting</td>
			<td>59020</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Stereotaxic Frame</td>
			<td>&nbsp;</td>
			<td>Stoeling</td>
			<td>51725</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Living Image Software</td>
			<td>&nbsp;</td>
			<td>Caliper Life Science</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>D-luciferin</td>
			<td>&nbsp;</td>
			<td>Gold Biotechnology</td>
			<td>LUCK-1G</td>
			<td>Potassium Salt</td>
		</tr>
		<tr>
			<td>Xenogen Lumina</td>
			<td>&nbsp;</td>
			<td>Caliper Life Science</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

establishing intracranial brain tumor xenografts with subsequent analysis of tumor growth and response to therapy using bioluminescence imaging

使用人脑肿瘤细胞的移植模型，有担任多年在神经肿瘤学研究的重要功能。在过去，最常用的程序，包括为人类肿瘤异种移植建立的细胞培养瓶中收集，收集细胞免疫功能低下小鼠皮下注射。鉴于这种做法仍然认为在许多实验室的频繁使用，出现了一个强调在过去十年中的重大转变，对原位移植瘤的建立，其中，在脑肿瘤的实例，需要到相应的神经解剖结构的肿瘤细胞注射。由于颅内移植瘤的建立，消除了监控能力通过直接测量肿瘤的生长，如使用卡钳，原位脑肿瘤异种移植模型中的重点转向转变，有必要利用的非侵入性的成像的评估宿主动物的肿瘤负担。目前可用的成像方法，被普遍认为是生物发光监测提供了灵敏度，权宜之计，和成本的最佳组合。在这里，我们将展示原位脑肿瘤的建立，为监测肿瘤的生长和实验疗法对治疗的反应时测试的程序。

Watch this Scientific Journal Video about Establishing Intracranial Brain Tumor Xenografts With Subsequent Analysis of Tumor Growth and Response to Therapy using Bioluminescence Imaging at JoVE.com

Establishing Intracranial Brain Tumor Xenografts With Subsequent Analysis of Tumor Growth and Response to Therapy using Bioluminescence Imaging

可以建立荧光素酶修饰人脑肿瘤异种移植，颅内无胸腺小鼠，随后的监测肿瘤的生长和治疗用生物发光成像。在与生存分析相结合，生物发光监测是一个重要的研究工具疗法被认为是治疗脑肿瘤的临床前测试。

建立颅内脑肿瘤异种移植肿瘤的生长和对治疗的反应与后续利用生物发光成像分析

Transplantation models using human brain tumor cells have served an essential function in neuro-oncology research for many years. In the past, the most ...

establishing-intracranial-brain-tumor-xenografts-with-subsequent

Research

JoVE Journal

Neuroscience

39.0K 次观看.  University of California, San Francisco - UCSF. 可以建立荧光素酶修饰人脑肿瘤异种移植，颅内无胸腺小鼠，随后的监测肿瘤的生长和治疗用生物发光成像。在与生存分析相结合，生物发光监测是一个重要的研究工具疗法被认为是治疗脑肿瘤的临床前测试。

视频: Establishing Intracranial Brain Tumor Xenografts With Subsequent Analysis of Tumor Growth and Response to Therapy using Bioluminescence Imaging

Brain Imaging Investigation of the Impairing Effect of Emotion on Cognition

Emotions can impact cognition by exerting both enhancing (e.g., better memory for emotional events) and impairing (e.g., increased emotional distractibility) effects (reviewed in 1). Complementing our recent protocol 2 describing a method that allows investigation of the neural correlates of the memory-enhancing effect of emotion (see also 1, 3-5), here we present a protocol that allows investigation of the neural correlates of the detrimental impact of emotion on cognition. The main feature of this method is that it allows identification of reciprocal modulations between activity in a ventral neural system, involved in 'hot' emotion processing (HotEmo system), and a dorsal system, involved in higher-level 'cold' cognitive/executive processing (ColdEx system), which are linked to cognitive performance and to individual variations in behavior (reviewed in 1). Since its initial introduction 6, this design has proven particularly versatile and influential in the elucidation of various aspects concerning the neural correlates of the detrimental impact of emotional distraction on cognition, with a focus on working memory (WM), and of coping with such distraction 7,11, in both healthy 8-11 and clinical participants 12-14.

We present a protocol that allows investigation of the neural mechanisms mediating the detrimental impact of emotion on cognition, using functional magnetic resonance imaging. This protocol can be used with both healthy and clinical participants.

脑成像的情感对认知损害的影响调查

Emotions can impact cognition by exerting both enhancing (e.g., better memory for emotional events) and impairing (e.g., increased emotional ...

科研

神经科学

Enabling High Grayscale Resolution Displays and Accurate Response Time Measurements on Conventional Computers

Display systems based on conventional computer graphics cards are capable of generating images with 8-bit gray level resolution. However, most experiments in vision research require displays with more than 12 bits of luminance resolution. Several solutions are available. Bit++ 1 and DataPixx 2 use the Digital Visual Interface (DVI) output from graphics cards and high resolution (14 or 16-bit) digital-to-analog converters to drive analog display devices. The VideoSwitcher 3 described here combines analog video signals from the red and blue channels of graphics cards with different weights using a passive resister network 4 and an active circuit to deliver identical video signals to the three channels of color monitors. The method provides an inexpensive way to enable high-resolution monochromatic displays using conventional graphics cards and analog monitors. It can also provide trigger signals that can be used to mark stimulus onsets, making it easy to synchronize visual displays with physiological recordings or response time measurements.
Although computer keyboards and mice are frequently used in measuring response times (RT), the accuracy of these measurements is quite low. The RTbox is a specialized hardware and software solution for accurate RT measurements. Connected to the host computer through a USB connection, the driver of the RTbox is compatible with all conventional operating systems. It uses a microprocessor and high-resolution clock to record the identities and timing of button events, which are buffered until the host computer retrieves them. The recorded button events are not affected by potential timing uncertainties or biases associated with data transmission and processing in the host computer. The asynchronous storage greatly simplifies the design of user programs. Several methods are available to synchronize the clocks of the RTbox and the host computer. The RTbox can also receive external triggers and be used to measure RT with respect to external events. 
Both VideoSwitcher and RTbox are available for users to purchase. The relevant information and many demonstration programs can be found at <a href="http://lobes.usc.edu/">http://lobes.usc.edu/</a>.

Conventional computer hardware can not generate visual stimuli with sufficiently high grayscale resolution and measure response times with sufficient accuracy. We describe how to use the VideoSwitcher to produce high-resolution monochromatic displays, and the RTbox to measure response times with high accuracy on conventional computer hardware.

启用高灰度分辨率的显示器和传统计算机准确的响应时间测量

Display systems based on conventional computer graphics cards are capable of generating images with 8-bit gray level resolution. However, most experiments ...

Isolation and Expansion of Human Glioblastoma Multiforme Tumor Cells Using the Neurosphere Assay

Stem-like cells have been isolated in tumors such as breast, lung, colon, prostate and brain. A critical issue in all these tumors, especially in glioblastoma mutliforme (GBM), is to identify and isolate tumor initiating cell population(s) to investigate their role in tumor formation, progression, and recurrence. Understanding tumor initiating cell populations will provide clues to finding effective therapeutic approaches for these tumors. The neurosphere assay (NSA) due to its simplicity and reproducibility has been used as the method of choice for isolation and propagation of many of this tumor cells. This protocol demonstrates the neurosphere culture method to isolate and expand stem-like cells in surgically resected human GBM tumor tissue. The procedures include an initial chemical digestion and mechanical dissociation of tumor tissue, and subsequently plating the resulting single cell suspension in NSA culture. After 7-10 days, primary neurospheres of 150-200 &mu;m in diameter can be observed and are ready for further passaging and expansion.

This video protocol demonstrates the isolation and expansion of stem like cells from surgically resected human glioblastoma mutliforme (GBM) tumor tissue using the neurosphere assay culture method.

人类胶质母细胞瘤肿瘤细胞的分离和扩大使用的神经球含量

Stem-like cells have been isolated in tumors such as breast, lung, colon, prostate and brain. A critical issue in all these tumors, especially in ...

Co-analysis of Brain Structure and Function using fMRI and Diffusion-weighted Imaging

The study of complex computational systems is facilitated by network maps, such as circuit diagrams. Such mapping is particularly informative when studying the brain, as the functional role that a brain area fulfills may be largely defined by its connections to other brain areas. In this report, we describe a novel, non-invasive approach for relating brain structure and function using magnetic resonance imaging (MRI). This approach, a combination of structural imaging of long-range fiber connections and functional imaging data, is illustrated in two distinct cognitive domains, visual attention and face perception. Structural imaging is performed with diffusion-weighted imaging (DWI) and fiber tractography, which track the diffusion of water molecules along white-matter fiber tracts in the brain (Figure 1). By visualizing these fiber tracts, we are able to investigate the long-range connective architecture of the brain. The results compare favorably with one of the most widely-used techniques in DWI, diffusion tensor imaging (DTI). DTI is unable to resolve complex configurations of fiber tracts, limiting its utility for constructing detailed, anatomically-informed models of brain function. In contrast, our analyses reproduce known neuroanatomy with precision and accuracy. This advantage is partly due to data acquisition procedures: while many DTI protocols measure diffusion in a small number of directions (e.g., 6 or 12), we employ a diffusion spectrum imaging (DSI)1, 2 protocol which assesses diffusion in 257 directions and at a range of magnetic gradient strengths. Moreover, DSI data allow us to use more sophisticated methods for reconstructing acquired data. In two experiments (visual attention and face perception), tractography reveals that co-active areas of the human brain are anatomically connected, supporting extant hypotheses that they form functional networks. DWI allows us to create a "circuit diagram" and reproduce it on an individual-subject basis, for the purpose of monitoring task-relevant brain activity in networks of interest.

We describe a novel approach for simultaneous analysis of brain function and structure using magnetic resonance imaging (MRI). We assess brain structure with high-resolution diffusion-weighted imaging and white-matter fiber tractography. Unlike standard structural MRI, these techniques allow us to directly relate anatomical connectivity to functional properties of brain networks.

使用功能磁共振成像和弥散加权成像脑结构与功能分析

The study of complex computational systems is facilitated by network maps, such as circuit diagrams. Such mapping is particularly informative when ...

Lineage-reprogramming of Pericyte-derived Cells of the Adult Human Brain into Induced Neurons

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis and enable new strategies for in vitro modeling or repairing the diseased brain. Identifying brain-resident non-neuronal cell types amenable to direct conversion into iNs might allow for launching such an approach in situ, i.e. within the damaged brain tissue. Here we describe a protocol developed in the attempt of identifying cells derived from the adult human brain that fulfill this premise. This protocol involves: (1) the culturing of human cells from the cerebral cortex obtained from adult human brain biopsies; (2) the in vitro expansion (approximately requiring 2-4 weeks) and characterization of the culture by immunocytochemistry and flow cytometry; (3) the enrichment by fluorescence-activated cell sorting (FACS) using anti-PDGF receptor-&#946; and anti-CD146 antibodies; (4) the retrovirus-mediated transduction with the neurogenic transcription factors sox2 and ascl1; (5) and finally the characterization of the resultant pericyte-derived induced neurons (PdiNs) by immunocytochemistry (14 days to 8 weeks following retroviral transduction). At this stage, iNs can be probed for their electrical properties by patch-clamp recording. This protocol provides a highly reproducible procedure for the in vitro lineage conversion of brain-resident pericytes into functional human iNs.

Targeting brain-resident cells for direct lineage-reprogramming offers new perspectives for brain repair. Here we describe a protocol of how to prepare cultures enriched for brain-resident pericytes from the adult human cerebral cortex and convert these into induced neurons by retrovirus-mediated expression of the transcription factors Sox2 and Ascl1.

谱系重编程的成人大脑的周细胞源性细胞诱导成神经细胞

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis ...

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible are badly known. Excitotoxicity, a process believed to contribute to neuron damage induced by both insults, is mediated by activation of glutamate receptors that promotes Ca2+ influx and mitochondrial Ca2+ overload. A substantial change in intracellular Ca2+ homeostasis or remodeling of intracellular Ca2+ homeostasis may favor neuron damage in old neurons. For investigating Ca2+ remodeling in aging we have used live cell imaging in long-term cultures of rat hippocampal neurons that resemble in some aspects aged neurons in vivo. For this end, hippocampal cells are, in first place, freshly dispersed from new born rat hippocampi and plated on poli-D-lysine coated, glass coverslips. Then cultures are kept in controlled media for several days or several weeks for investigating young and old neurons, respectively. Second, cultured neurons are loaded with fura2 and subjected to measurements of cytosolic Ca2+ concentration using digital fluorescence ratio imaging. Third, cultured neurons are transfected with plasmids expressing a tandem of low-affinity aequorin and GFP targeted to mitochondria. After 24 hr, aequorin inside cells is reconstituted with coelenterazine and neurons are subjected to bioluminescence imaging for monitoring of mitochondrial Ca2+ concentration. This three-step procedure allows the monitoring of cytosolic and mitochondrial Ca2+ responses to relevant stimuli as for example the glutamate receptor agonist NMDA and compare whether these and other responses are influenced by aging. This procedure may yield new insights as to how aging influence cytosolic and mitochondrial Ca2+ responses to selected stimuli as well as the testing of selected drugs aimed at preventing neuron cell death in age-related diseases.

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Intracellular Ca2+ remodeling in aging may contribute to excitotoxicity and neuron damage, processes mediated by Ca2+ overload. We aimed at investigating Ca2+ remodeling in the aging brain using fluorescence and bioluminescence imaging of cytosolic and mitochondrial Ca2+ in long-term cultures of rat hippocampal neurons, a model of neuronal aging.

亚细胞钙荧光和生物发光成像<sup&gt; 2+</sup&gt;老年海马神经元

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible ...

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 ...

Peering at Brain Polysomes with Atomic Force Microscopy

The translational machinery, i.e., the polysome or polyribosome, is one of the biggest and most complex cytoplasmic machineries in cells. Polysomes, formed by ribosomes, mRNAs, several proteins and non-coding RNAs, represent integrated platforms where translational controls take place. However, while the ribosome has been widely studied, the organization of polysomes is still lacking comprehensive understanding. Thus much effort is required in order to elucidate polysome organization and any novel mechanism of translational control that may be embedded. Atomic force microscopy (AFM) is a type of scanning probe microscopy that allows the acquisition of 3D images at nanoscale resolution. Compared to electron microscopy (EM) techniques, one of the main advantages of AFM is that it can acquire thousands of images both in air and in solution, enabling the sample to be maintained under near physiological conditions without any need for staining and fixing procedures. Here, a detailed protocol for the accurate purification of polysomes from mouse brain and their deposition on mica substrates is described. This protocol enables polysome imaging in air and liquid with AFM and their reconstruction as three-dimensional objects. Complementary to cryo-electron microscopy (cryo-EM), the proposed method can be conveniently used for systematically analyzing polysomes and studying their organization.

While ribosome structure has been extensively characterized, the organization of polysomes is still understudied. To overcome this lack of knowledge, we present here a detailed preparation protocol for accurate imaging of mammalian polysomes by atomic force microscopy (AFM) in air and liquid.

在脑核糖凝视与原子力显微镜

The translational machinery, i.e., the polysome or polyribosome, is one of the biggest and most complex cytoplasmic machineries in cells. Polysomes, ...

Kinematic Analysis Using 3D Motion Capture of Drinking Task in People With and Without Upper-extremity Impairments

Kinematic analysis is a powerful method for objective assessment of upper extremity movements in a three-dimensional (3D) space. Three-dimensional motion capture with an optoelectronic camera system is considered as golden standard for kinematic movement analysis and is increasingly used as outcome measure to evaluate the movement performance and quality after an injury or disease involving upper extremity movements. This article describes a standardized protocol for kinematic analysis of drinking task applied in individuals with upper extremity impairments after stroke. The drinking task incorporates reaching, grasping and lifting a cup from a table to take a drink, placing the cup back, and moving the hand back to the edge of the table. The sitting position is standardized to the individual&#39;s body size and the task is performed in a comfortable self-paced speed and compensatory movements are not constrained. The intention is to keep the task natural and close to a real-life situation to improve the ecological validity of the protocol. A 5-camera motion capture system is used to gather 3D coordinate positions from 9 retroreflective markers positioned on anatomical landmarks of the arm, trunk, and face. A simple single marker placement is used to ensure the feasibility of the protocol in clinical settings. Custom-made Matlab software provides automated and fast analyses of movement data. Temporal kinematics of movement time, velocity, peak velocity, time of peak velocity, and smoothness (number of movement units) along with spatial angular kinematics of shoulder and elbow joint as well as trunk movements are calculated. The drinking task is a valid assessment for individuals with moderate and mild upper extremity impairment. The construct, discriminative and concurrent validity along with responsiveness (sensitivity to change) of the kinematic variables obtained from the drinking task have been established.

This protocol describes an objective method to evaluate the movement performance and sensorimotor function of the upper extremity applied to individuals with stroke and healthy controls. A standardized test procedure, kinematic analysis and outcome variables for three-dimensional motion capture of drinking task are provided.

用3D 运动捕捉对无上肢损伤患者饮酒任务的运动学分析

Kinematic analysis is a powerful method for objective assessment of upper extremity movements in a three-dimensional (3D) space. Three-dimensional motion ...

Analyzing Neural Activity and Connectivity Using Intracranial EEG Data with SPM Software

Measuring neural activity and connectivity associated with cognitive functions at high spatial and temporal resolutions is an important goal in cognitive neuroscience. Intracranial electroencephalography (EEG) can directly record electrical neural activity and has the unique potential to accomplish this goal. Traditionally, averaging analysis has been applied to analyze intracranial EEG data; however, several new techniques are available for depicting neural activity and intra- and inter-regional connectivity. Here, we introduce two analytical protocols we recently applied to analyze intracranial EEG data using the Statistical Parametric Mapping (SPM) software: time-frequency SPM analysis for neural activity and dynamic causal modeling of induced responses for intra- and inter-regional connectivity. We report our analysis of intracranial EEG data during the observation of faces as representative results. The results revealed that the inferior occipital gyrus (IOG) showed gamma-band activity at very early stages (110 ms) in response to faces, and both the IOG and amygdala showed rapid intra- and inter-regional connectivity using various types of oscillations. These analytical protocols have the potential to identify the neural mechanisms underlying cognitive functions with high spatial and temporal profiles.

We present two analytical protocols that can be used to analyze intracranial electroencephalography data using the Statistical Parametric Mapping (SPM) software: time-frequency statistical parametric mapping analysis for neural activity, and dynamic causal modeling of induced responses for intra- and inter-regional connectivity.

利用 SPM 软件分析颅内脑电图数据的神经活动和连接性

Measuring neural activity and connectivity associated with cognitive functions at high spatial and temporal resolutions is an important goal in cognitive ...