朱庇特的视频文件的支持，这项工作是由美国国立卫生研究院NINDS授予R01 NS074388 581（CL Limoli），美国加州再生医学研究所（CIRM）批准RS1 - 00413（CLL）和CIRM格兰特支持。我们还要感谢他们的意见，支持和精湛的技术技能，行为测试中使用的所有定制的材料准备的鲁迪 - 林堡和爱德华美国加州大学欧文分校的生物科学机械厂刘。

我们的认知测试范例示意图图示于图1 。认知测试开始照射后1个月，手术过程中进行。 进行新的地点识别（NPR）的任务<ol class="lalpha"><li>小说广场识别（NPR）的任务是根据Mumby和他的同事（2002）所描述的程序，是一个空间识别记忆任务依赖于海马4。 </li><li>大鼠是第一次熟悉与测试环境中，放置在测试场馆三天每天一次20分钟（定制，白色，丙烯酸，打开机顶盒，70厘米X70厘米X 40厘米高）。这些习惯会议期间，不会对以后的测试中使用的对象被放置在赛场。 </li><li>对于习惯阶段，应该指出的探索行为的人工观测，同时记录和跟踪的行为是没有必要的。 </li><li>对于核态势评估任务的各个阶段，赛场彻底清洗，使用70％的乙醇，以减少混杂的气味线索的动物之间。对于所有的试验中，实验者在方向一致的测试舞台的中心，以尽量减少动物的机会，动物只会探索，他们碰巧被放在旁边的块。 </li><li>熟悉阶段和5分钟的测试阶段，管理上​​的第四天。 </li><li>对于熟悉阶段，两个相同的塑料块（8CM X3厘米x 10厘米高），已用70％乙醇彻底清洗的被放置在测试场馆内的具体位置。一个动物是摆在每个舞台上5分钟，并允许自由探索。 </li><li>使用自动跟踪​​系统的探索取得了同样熟悉的两个空间位置。所有阶段，勘探是指动物的鼻子点一道4厘米半径内和朝块。 </li><li>对于llowing熟悉阶段，动物被放置在一个小的控股笼延迟间隔为5分钟。在这段时间内，测试，阿里纳斯是用70％乙醇清洗，并添加块完全相同的副本;在熟悉阶段在相同的空间位置，并在一个新的空间位置。如果一只老鼠记得从熟悉阶段块的空间安排，将花费更多的时间探索小说中的空间位置位于块。 </li><li>经过5分钟的延迟间隔，动物测试，阿里纳斯为5分钟的测试阶段。他们自由探索为3分钟。 </li><li>熟悉阶段结束后24小时内，动物返回测试，阿里纳斯再次为24小时的测试阶段。在这一阶段，一个块仍然在相同的位置，在熟悉和5分钟的测试阶段，和其他块被放置在一个新的，新颖的空间位置。同样，如果一只老鼠重新块的前一天从熟悉阶段的空间安排的成员，他将花更多的时间探索小说中的空间位置位于块。 </li><li>现在，探索在不同空间位置的块所花费的时间是使用自动跟踪​​程序收集，可以分析实验组之间的差异有统计学。 </li><li>对于熟悉阶段，花费的总时间探索整个审判的空间位置计算。应该有没有探索在这个阶段的两个空间位置所花费的时间差，因为他们同样熟悉/陌生的动物（图2A）。此外，实验组之间的总勘探显著的差异可能表明在探索的动机或焦虑水平变化，并解释从两个测试阶段空间识别记忆的结果时，应考虑。 </li><li>对于这两种5分钟和24小时的测试阶段，勘探比率计算如下:所花费的时间探索小说的立场-探索熟悉的位置上徜徉（ 图2B）/总时间花费的时间。这样一来，在探索对象的动机个体差异考虑在内。正常动物花更多的时间，探索小说的空间位置（虚线表示）的机会比预期后，5分钟和24小时的延迟时间间隔（图2B）。 </li><li>通常是探索的比例计算，5分钟和24小时的测试4第60秒。这是因为，作为检验审判的进展情况，新颖的空间位置变得更加熟悉的动物。因此，第一分钟的测试试验提供了空间识别记忆的最准确的评估。 </li></ol> 2。进行高架十字迷宫（EPM）的任务<ol class="lalpha"><li>高架十字迷宫（EPM）任务的基础上，2007年5 Walf和弗莱的程序。 </li><li> EPM任务是用来评估在啮齿类动物的焦虑。 EPM仪器4迷宫（45"× 4"）提出的45"从地面水平，在加的形状排列的武器。两只手臂封闭和开放。表明该仪器是医学同仁公司制造。 </li><li>在此任务的行​​为，反映了老鼠的天然动机，探索新的环境张开双臂所花费的时间和留在保护区（封闭式武器）偏好之间的冲突。 </li><li>在迷宫中指出的射灯，以确保有足够的动机，喜欢封闭，光线昏暗，武器试验室灯火通明。 </li><li>迷宫彻底清洗前的动物试验，每次试验之间。 </li><li>动物是放置在仪器下前肢，并放置在迷宫的中心部分，面临着开放的手臂，是远离实验者。每个试验持续时间为5分钟。 </li><li>动物的路径是这样的条目，并在开放式和封闭式的武器所花费的时间金额数目可以计算使用自动跟踪​​软件跟踪。其他行为，如在仪器侧面的养育，培养和头部逢低可以监视。当动物的中心点到一条胳膊穿过一个条目计数。 </li><li>如果一只老鼠脱落的迷宫，它正在迅速放回开放的手臂从它下跌，而该事件在观察笔记本记录。如果一只老鼠长时间冻结的行为，以及任何可能的原因（ 如突然，试验室以外的巨响）指出，和测试继续使所有实验动物也有类似的曝光时间。 </li><li>在开放与封闭的武器花费的时间是用来计算比例:张开双臂/武器的总时间。同样，打开条目的比例与总的武器ARM项计算。使用自动跟踪​​软件，总行驶距离和速度也可以测量，以评估在实验组之间的运动功能的任何分歧。 </li><li>减少焦虑与更大比例的时间花费在张开双臂，而花更多的时间在封闭的武器增加了焦虑和恐惧。正常动物花张开双臂显著较少的时间比预期的机会（由虚线，图 3表示） 。更着急的动物在张开双臂将花费更少的时间比例，而较少焦虑的动物会花更多的时间在张开双臂（例如，在迷宫或以下的抗焦虑药物治疗的多重曝光）。 </li></ol> 3。在进行背景和线索恐惧制约（FC）的任务<ol class="lalpha"><li>背景和线索恐惧制约（下称"财委会"）的任务描述的基础上，从Schaf程序E等人，1999年，Winocur等人，2006年和MacLeod等人，2007年6-8。 </li><li>恐惧制约（下称"财委会"）的任务是在动物学会害怕先前的中性刺激（背景下，光或音;条件刺激）配对有害刺激（通常是一个温和的电击条件刺激的行为范式）。学习是快速，健壮和长久的，因此FC的任务是经常使用的研究在啮齿类动物学习和记忆的神经生物学电路。 </li><li> FC设备是一个30厘米x 30厘米x 40cm高的封闭室（Phenotyper，型号3000，Noldus信息技术）。室的地板由一个不锈钢网，这是用来提供电击刺激。 Phenotyper是配备了一个扬声器发出2300Hz，需求90分贝音，作为条件刺激。 </li><li>通过冲击发生器（MED Associates公司）交付的震撼。休克是一时的不适，这不会产生任何拉斯维加斯婷疼痛或组织损伤。培训和测试试验，交付的冲击和音刺激定时和控制自动使用的Ethovision XT添加试验和硬件控制模块（版本7.0; Noldus信息技术）。 </li><li>在第一天，FC进行培训。 Phenotyper是用70％乙醇，彻底清洗和一个单一的动物，是放置在会议厅内。培训试验分为三个阶段:（1）基线阶段，没有休克或音管理，（2）训练阶段，当音休克配对，和（3）培训后阶段，当无休克或音管理。每个阶段持续约5分钟，共培训试验动物每15分钟的时间。 </li><li>在训练阶段，管理的基调是30秒和一致的基调结束，1毫安冲击传递到网格为1秒。这口气休克配对重复了5次，一个​​变量的10至60塞科ND每个配对之间的间隔间距。 </li><li>从训练阶段，每个动物花费时间的百分比一动不动，或冻结，测量是从最后60秒的初始基线阶段（之前建立的基调休克配对自发冻结），并从最后60秒后训练阶段（建立在基调休克对冻结行为）。正常动物显示非常低的水平冻结在基线阶段和非常高（〜95％）在培训后的阶段（ 图4）冻结水平。 </li><li>动物24小时后，返回到相同的情况下，观察5分钟的上下文功能界别测试。在这个阶段，无休克或音交付，冻结的百分比，所花费的时间是整个审判计算。注意保持恒定的范围内，从培训到上下文测试的重要性。事实上，有相同的实验者处理和试验动物，这两天是NEcessary。正常动物的花费约70％从事冻结行为（ 图4）的范围内试验。 </li><li>一小时后的线索FC测试管理。室中删除格地板，板室外面，带有香味的家用清洁剂和清洁地板的改变。冻结动物的行为是观察的第一分钟，并冻结水平非常低，如果上下文得到了充分的改变（图4）。的语气，然后打了3分钟，最后一分钟的沉默，并花了冻结计算的百分比时间。正常动物显示（ 图4）在此期间，高层次的冻结。 </li><li>共收集了5冻结行为的离散措施;（1）基线;（2）岗位培训;（3）范围内试验;（4）预先提示测试;（5）提示和线索后的测试。 </li><li>在基线阶段冻结的情况下建立正常的焦虑水平，而实质上TiAl基冻结在培训后阶段建议完好的感觉运动能力。冻结上下文测试期间建立24小时内存训练的情况下，依靠完整的海马和杏仁核的功能9。缺乏预先提示测试冻结初始培训方面的建议足够的改建。 Cue和冻结线索后，建立24小时内存为基调的刺激下，依靠完整的杏仁核功能9。 </li></ol> 4。进行Morris水迷宫（MWM）任务<ol class="lalpha"><li> Morris水迷宫任务的程序是基于10,11以前的描述。 </li><li> Morris水迷宫（MWM）任务，需要使用远端线索定位在一个不透明的水大池的水下逃生平台的啮齿动物的空间学习测试。周围放置在特定的，恒定的位置池大，不同的视觉线索。动物学会利用这些线索，导航到苏bmerged平台。动物进行监测，并从水中取出，如果他们不能游泳。 </li><li>跨反复的试验和评估偏爱的平台区，平台是缺席时确定参考记忆空间学习评估。逆转试验提高检测的空间障碍，并可能税附加的大脑区域如额叶皮层 10 。 </li><li> MWM的任务是管理超过8天，包括4个阶段;一个线索的测试，采集阶段，一个24小时的探针测试和逆转学习测试。 </li><li>池是一个特制的白色，聚乙烯罐，200厘米直径和60厘米高。它是装满了水，深约30厘米和不透明的白色，无毒漆。保持水温20日至22 ° C（68和72 ° F）。逃生平台是一个特制的，一个圆形的顶部测量直径10cm的透明塑料立场。它位于WATE表面以下约1.5厘米河</li><li>线索测试包括4项试验，第1天给药。对于这些试验中，一个鲜明的黑色和白色标志，是摆在中间的平台，使逃生位置是明显的动物的。为了尽量减少使用的空间信息，在此阶段，墙上的线索和平台，并开始地点是多种多样的审判审判。这个阶段有助于排除实验组之间的任何感觉运动有关的差异，收购前阶段开始。 </li><li> MWM任务的各个阶段，动物前肢下举行，在水中轻轻地放在指定的开始位置，面对池壁。动物测试等8个，有8至10分钟审间每日4试验之间的间隔的同伙。审间的间隔期间，动物在变暖笼，以避免低温保存。 </li><li>线索测试，最多为30秒游泳时间允许为动物爬上平台。如果动物逃脱之前达到最大的时间是，实验者指导平台手放在平台上。动物的尸体为一个20秒的休息时间，被放置在一个变暖笼前的平台。 </li><li>对于每一个线索测试试验，动物开始在池周边的不同，预先确定的起始位置，平台也是移动的4个不同的象限位置。 </li><li>此次收购阶段就开始了次日连续5天每天4个试验组成。在这个阶段，隐藏的平台仍然在相同的位置和动物学习来找到它使用远端房间的线索。 </li><li>同样，每个4项试验，动物在池中放置在一个不同的，预先确定的起点。每一天，两个起点是左侧的两个出发点是该平台的权利，以随机顺序，避免egoc发展entric空间的战略要解决的任务。 </li><li>每个收购审判的最大的游泳时间是60秒，后动物是引导和放置在平台上的实验者。动物的尸体为20秒以下转义的平台上。 </li><li>第7天，24小时后的最后一次训练试验，其中一个60秒的探测试验管理平台被删除。这个阶段决定动物的偏好象限包含的平台，是用于衡量空间参考记忆10。探头试验之后，返回到同一象限，3定期采集试验平台管理。 </li><li>在第8天的学习测试逆转，平台移动到一个新的位置，以评估逆转学习能力。测量动物的时间量花在寻找在它与新的位置上位置的平台，提供perseveration评估，或INAbility压制响应一次平台上的位置已经移到 10 。至于收购和探测阶段，4个60秒的试验管理。 </li><li>线索测试，找到平台和速度的延迟作为视觉和运动功能的措施，分别。在这个阶段的实验组之间的差异，必须考虑在群体之间的感觉运动功能差异可能存在的收购和内存结果的解释。 </li><li>收购阶段，平均潜伏期找到平台和/或路径长度为空间学习的措施。在正常动物，逃避潜伏期逐渐跌幅超过五个收购天平均逃逸时间约15至20秒第5天（ 图5A）。 </li><li>探测阶段，意味着平台的距离（ 图5B）和/或延迟首先到达平台的先前的位置（ 图 5C </强&gt;）作为空间参考记忆的措施。 </li><li>逆转阶段，％的时间花费在与新老象限包含平台是用来作为衡量一个perseveration。四项试验中，为老平台的位置应优先减少偏爱新平台的位置（ 图5D）。 </li></ol> 5。代表性的成果: <img alt="图1" src="/files/ftp_upload/3108/3108fig1.jpg" /> 图1:我们的认知测试范例示意图。 <img alt="图2" src="/files/ftp_upload/3108/3108fig2.jpg" /> 图2代表从控制动物小说的地方识别任务。一）探索两个空间位置花费的总时间应在熟悉阶段大致相等。 b）探讨比率（时间花在探索新颖的宝sition /花时间探索这两个职位），显示了明显的偏好小说的空间位置后，5分钟和24小时的延迟间隔。虚线表示有机会表现。 <img alt="图3" src="/files/ftp_upload/3108/3108fig3.jpg" /> 图3从控制动物的代表对高架十字迷宫。正常动物花费更少的时间比预期在张开双臂（虚线表示）的机会。焦急的动物会花时间在张开双臂，与对照组相比比例少，而多重曝光的迷宫或抗焦虑剂治疗会增加花费的时间在张开双臂。 <img alt="图4" src="/files/ftp_upload/3108/3108fig4.jpg" /> 图4:从控制动物的代表担心空调任务。观察期间初次接触室（基线水平低的冻结），当上下文改变之前提示测试（前提示测试）。相比之下，正常动物花费大部分从事冻结行为的培训后的时期。如果动物记得的背景下发生后24小时的延迟，其中培训的，他们将进行5分钟的范围内试验约70％的冻结行为。同样，如果动物记住的基调，他们的提示和线索后测试表明，在高层次冻结。 <img alt="图5" src="/files/ftp_upload/3108/3108fig5.jpg" /> 图5。Morris水迷宫任务控制动物的代表结果。一）在收购阶段，循序渐进的训练逃避潜伏期下降。 b）和c）平均距离和延迟，第一跨在24小时探头测试前面的平台位置，作为空间参考记忆的措施。四）逆转学习评估平台的位置改变的工商业污水附加费的最后一天婷。 4项试验，正常动物逐渐停止寻找老象限和学习，该平台是一个新的位置。

<ol>
	<li>Abayomi, O. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+irradiation-induced+cognitive+dysfunction.">Pathogenesis of irradiation-induced cognitive dysfunction.</a> Acta. Oncol. 35, 659-663 (1996).</li><li>Anderson, V. A., Godber, T., Smibert, E., Weiskop, S., Ekert, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cognitive+and+academic+outcome+following+cranial+irradiation+and+chemotherapy+in+children:+a+longitudinal+study.">Cognitive and academic outcome following cranial irradiation and chemotherapy in children: a longitudinal study.</a> Br. J. Cancer. 82, 255-262 (2000).</li><li>Acharya, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rescue+of+radiation-induced+cognitive+impairment+through+cranial+transplantation+of+human+embryonic+stem+cells.">Rescue of radiation-induced cognitive impairment through cranial transplantation of human embryonic stem cells.</a> Proc. Natl. Acad. Sci. U. S. A. 106, 19150-19155 (2009).</li><li>Mumby, D. G., Gaskin, S., Glenn, M. J., Schramek, T. E., Lehmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hippocampal+damage+and+exploratory+preferences+in+rats:+memory+for+objects,+places,+and+contexts.">Hippocampal damage and exploratory preferences in rats: memory for objects, places, and contexts.</a> Learn. Mem. 9, 49-57 (2002).</li><li>Walf, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+the+elevated+plus+maze+as+an+assay+of+anxiety-related+behavior+in+rodents.">The use of the elevated plus maze as an assay of anxiety-related behavior in rodents.</a> Nat. Protoc. 2, 322-328 (2007).</li><li>Macleod, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+chemotherapy+impairs+contextual+but+not+cue-specific+fear+memory.">Cancer chemotherapy impairs contextual but not cue-specific fear memory.</a> Behav Brain Res. 181, 168-172 (2007).</li><li>Schafe, G. E., Nadel, N. V., Sullivan, G. M., Harris, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Memory+consolidation+for+contextual+and+auditory+fear+conditioning+is+dependent+on+protein+synthesis,+PKA,+and+MAP+kinase.">Memory consolidation for contextual and auditory fear conditioning is dependent on protein synthesis, PKA, and MAP kinase.</a> Learn. Mem. 6, 97-110 (1999).</li><li>Winocur, G., Wojtowicz, J. M., Sekeres, M., Snyder, J. S., Wang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+neurogenesis+interferes+with+hippocampus-dependent+memory+function.">Inhibition of neurogenesis interferes with hippocampus-dependent memory function.</a> Hippocampus. 16, 296-304 (2006).</li><li>Phillips, R. G., LeDoux, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+contribution+of+amygdala+and+hippocampus+to+cued+and+contextual+fear+conditioning.">Differential contribution of amygdala and hippocampus to cued and contextual fear conditioning.</a> Behav. Neurosci. 106, 274-285 (1992).</li><li>D'Hooge, R., De Deyn, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+the+Morris+water+maze+in+the+study+of+learning+and+memory.">Applications of the Morris water maze in the study of learning and memory.</a> Brain. Res. Brain. Res. Rev. 36, 60-90 (2001).</li><li>Morris, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developments+of+a+water-maze+procedure+for+studying+spatial+learning+in+the+rat.">Developments of a water-maze procedure for studying spatial learning in the rat.</a> J. Neurosci. Methods. 11, 47-60 (1984).</li><li>Meyers, C. A., Brown, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+and+relevance+of+neurocognitive+assessment+in+clinical+trials+of+patients+with+CNS+tumors.">Role and relevance of neurocognitive assessment in clinical trials of patients with CNS tumors.</a> J. Clin. Oncol. 24, 1305-1309 (2006).</li><li>de Ruiter, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebral+hyporesponsiveness+and+cognitive+impairment+10+years+after+chemotherapy+for+breast+cancer.">Cerebral hyporesponsiveness and cognitive impairment 10 years after chemotherapy for breast cancer.</a> Hum. Brain. Mapp. , (2010).</li><li>Hess, D. C., Borlongan, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-based+therapy+in+ischemic+stroke.">Cell-based therapy in ischemic stroke.</a> Expert. Rev. Neurother. 8, 1193-1201 (2008).</li></ol>

没有利益冲突的声明。

迫切需要有一个长期战略旨在防止认知功能障碍的发展，正在成为越来越常见癌症幸存者12,13。干细胞疗法可能会提供纾缓在中枢神经系统的正常组织后遗症范围的手段，为他们提供机会为丢失或细胞毒性癌症治疗14损坏的更换细胞。要加快实现这些努力，我们已经采取了一系列已经证实颅嫁接hNSCs功能恢复内照射组织的中枢神经系统的3床的认知能力的主要实验证明。这一努力的关键是能力可靠地进行了一系列精心控制的认知测试，在重现的方式。在努力加快干细胞疗法在临床上的平移的潜力，我们有详细的这些关键的程序。进一步考虑下面突出显示可能有一个数据质量的显著影响。 认知测试要求认真注意细节，并坚持一定的标准做法，可以帮助减少对结果的解释复杂的工件。使用专用的行为检测室，动物饲养室和实验者单独是可取的。它也谨慎地使用"白色噪音"，阻止外部噪声可能分散在手头的任务动物在动物设施。为了尽量减少认知测试注定动物压力，重要的是让他们熟悉实验者将执行行为测试前的任务管理。在一般情况下，如果测试男性和女性，建议来测试女性第一，因为他们往往​​更急于在测试过程中，如果男性气味线索的测试，阿里纳斯仍然。管理的任务串行电池时，重要的是要揭露动物以LEAST（EPM一样，NPR）去年最紧张的任务（如FC，MWM）的压力任务。

quantifying cognitive decrements caused by cranial radiotherapy

随着生存异常，认知功能障碍，癌症的临床管理带来口述治疗效果的一个主要因素。对于与中枢神经系统和非中枢神经系统恶性肿瘤影响的许多患者，放疗和化疗提供用于疾病控制的最佳选择。然而，这些治疗方法的成本，和几乎所有癌症幸存者（〜11万美元在美国仅2006年）因颅放疗（〜20万/年的患者中发现的一些最严重的情况下发展认知功能障碍的风险， ）为控制原发性和转移性脑肿瘤1。问题尤其严重的是儿科的情况下，其长期生存与显着的认知递减结果显著的社会经济负担2困扰。迄今为止，仍然没有令人满意的解决办法，这个显著的临床问题。 我们已经解决了这个严重的健康问题，移植的干CELLS打击辐射诱发的认知下降无胸腺大鼠颅照射3。立体定向照射和在体外培养人类神经干细胞移植（hNSCs）的详情，可在我们的配套文件（阿查里雅等人，朱庇特参考）。照射和移植手术后，老鼠，然后评估认知的变化，移植细胞的存活和分化的特异性标志物1和照射后4个月表达。审慎评估任何潜在的干预，以减轻辐射引起的认知后遗症，必须执行一系列严格的定量的认知任务的成功或失败。而要做到这一点，我们的主题动物的认知测试范例，包括新颖的地方认同，水迷宫，高架十字迷宫和恐惧条件套件，以量化的海马和非海马的学习和记忆。我们已经证明的效用这些测试照射动物的认知递减的特定类型的量化，并用它们来显示，与hNSCs嫁接的动物表现出显著改善认知功能3。 从嫁接的人类干细胞的认知利益，建议类似的策略，也许有一天提供急需的临床诉诸患有癌症幸存者从受损的认知。因此，我们必须提供书面和视​​觉在我们的认知测试范例中使用的关键步骤，以方便我们希望的结果翻译成诊所的文档。

Watch this Scientific Journal Video about Quantifying Cognitive Decrements Caused by Cranial Radiotherapy at JoVE.com

Quantifying Cognitive Decrements Caused by Cranial Radiotherapy

从脑肿瘤放射治疗的管理而造成的认知功能障碍临床上棘手的状况产生不利影响生​​活质量。最终取决于能力进行严格的定量评估认知能力，审慎评估可能的干预措施，缓解辐射引起的认知递减。

量化颅放疗引起的认知递减

With the exception of survival, cognitive impairment stemming from the clinical management of cancer is a major factor dictating therapeutic outcome. For ...

quantifying-cognitive-decrements-caused-by-cranial-radiotherapy

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

12.6K 次观看.  University of California Irvine. 从脑肿瘤放射治疗的管理而造成的认知功能障碍临床上棘手的状况产生不利影响生​​活质量。最终取决于能力进行严格的定量评估认知能力，审慎评估可能的干预措施，缓解辐射引起的认知递减。

视频: Quantifying Cognitive Decrements Caused by Cranial Radiotherapy

Stem Cell Transplantation Strategies for the Restoration of Cognitive Dysfunction Caused by Cranial Radiotherapy

Radiotherapy often provides the only clinical recourse for those afflicted with primary or metastatic brain tumors. While beneficial, cranial irradiation can induce a progressive and debilitating decline in cognition that may, in part, be caused by the depletion of neural stem cells. Given the increased survival of patients diagnosed with brain cancer, quality of life in terms of cognitive health has become an increasing concern, especially in the absence of any satisfactory long-term treatments.
To address this serious health concern we have used stem cell replacement as a strategy to combat radiation-induced cognitive decline. Our model utilizes athymic nude rats subjected to cranial irradiation. The ionizing radiation is delivered as either whole brain or as a highly focused beam to the hippocampus via linear accelerator (LINAC) based stereotaxic radiosurgery. Two days following irradiation, human neural stem cells (hNSCs) were stereotaxically transplanted into the hippocampus. Rats were then assessed for changes in cognition, grafted cell survival and for the expression of differentiation-specific markers 1 and 4-months after irradiation. Our cognitive testing paradigms have demonstrated that animals engrafted with hNSCs exhibit significant improvements in cognitive function. Unbiased stereology reveals significant survival (10-40%) of the engrafted cells at 1 and 4-months after transplantation, dependent on the amount and type of cells grafted. Engrafted cells migrate extensively, differentiate along glial and neuronal lineages, and express a range of immature and mature phenotypic markers.
Our data demonstrate direct cognitive benefits derived from engrafted human stem cells, suggesting that this procedure may one day afford a promising strategy for the long-term functional restoration of cognition in individuals subjected to cranial radiotherapy. To promote the dissemination of the critical procedures necessary to replicate and extend our studies, we have provided written and visual documentation of several key steps in our experimental plan, with an emphasis on stereotaxic radiosurgey and transplantation.

Brain tumor patients routinely undergo cranial radiotherapy, and while beneficial, this treatment often results in debilitating cognitive dysfunction. This serious unresolved problem has at present, no clinical recourse, and has driven our efforts to devise stem cell based therapies for the recovery of radiation-induced cognitive decrements.

颅放疗引起的认知功能障碍恢复干细胞移植策略

Radiotherapy often provides the only clinical recourse for those afflicted with primary or metastatic brain tumors. While beneficial, cranial irradiation ...

科研

医学

Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the understanding of the mechanics of tumor invasion, and direct intracerebral inoculation of tumor provides the opportunity of observing the invasive process in a physiologically appropriate environment2. As far as human brain tumors are concerned, the orthotopic models currently available are established either by stereotaxic injection of cell suspensions or implantation of a solid piece of tumor through a complicated craniotomy procedure3. In our technique we harvest cells from tissue culture to create a cell suspension used to implant directly into the brain. The duration of the surgery is approximately 30 minutes, and as the mouse needs to be in a constant surgical plane, an injectable anesthetic is used. The mouse is placed in a stereotaxic jig made by Stoetling (figure 1). After the surgical area is cleaned and prepared, an incision is made; and the bregma is located to determine the location of the craniotomy. The location of the craniotomy is 2 mm to the right and 1 mm rostral to the bregma. The depth is 3 mm from the surface of the skull, and cells are injected at a rate of 2 &mu;l every 2 minutes. The skin is sutured with 5-0 PDS, and the mouse is allowed to wake up on a heating pad. From our experience, depending on the cell line, treatment can take place from 7-10 days after surgery. Drug delivery is dependent on the drug composition. For radiation treatment the mice are anesthetized, and put into a custom made jig. Lead covers the mouse's body and exposes only the brain of the mouse. The study of tumorigenesis and the evaluation of new therapies for GBM require accurate and reproducible brain tumor animal models. Thus we use this orthotopic brain model to study the interaction of the microenvironment of the brain and the tumor, to test the effectiveness of different therapeutic agents with and without radiation.

The purpose of this article is to describe the use of an orthotopic glioblastoma model for chemoradiation studies. This article will go though cell processing, implanting, and radiotherapy of the mouse using an intracranial model.

在原位小鼠脑肿瘤模型结合放疗

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the ...

High Content Screening in Neurodegenerative Diseases

The functional annotation of genomes, construction of molecular networks and novel drug target identification, are important challenges that need to be addressed as a matter of great urgency1-4. Multiple complementary 'omics' approaches have provided clues as to the genetic risk factors and pathogenic mechanisms underlying numerous neurodegenerative diseases, but most findings still require functional validation5. For example, a recent genome wide association study for Parkinson's Disease (PD), identified many new loci as risk factors for the disease, but the underlying causative variant(s) or pathogenic mechanism is not known6, 7. As each associated region can contain several genes, the functional evaluation of each of the genes on phenotypes associated with the disease, using traditional cell biology techniques would take too long. 
	There is also a need to understand the molecular networks that link genetic mutations to the phenotypes they cause. It is expected that disease phenotypes are the result of multiple interactions that have been disrupted. Reconstruction of these networks using traditional molecular methods would be time consuming. Moreover, network predictions from independent studies of individual components, the reductionism approach, will probably underestimate the network complexity8. This underestimation could, in part, explain the low success rate of drug approval due to undesirable or toxic side effects. Gaining a network perspective of disease related pathways using HT/HC cellular screening approaches, and identifying key nodes within these pathways, could lead to the identification of targets that are more suited for therapeutic intervention.
	High-throughput screening (HTS) is an ideal methodology to address these issues9-12. but traditional methods were one dimensional whole-well cell assays, that used simplistic readouts for complex biological processes. They were unable to simultaneously quantify the many phenotypes observed in neurodegenerative diseases such as axonal transport deficits or alterations in morphology properties13, 14. This approach could not be used to investigate the dynamic nature of cellular processes or pathogenic events that occur in a subset of cells. To quantify such features one has to move to multi-dimensional phenotypes termed high-content screening (HCS)4, 15-17. HCS is the cell-based quantification of several processes simultaneously, which provides a more detailed representation of the cellular response to various perturbations compared to HTS. 
	HCS has many advantages over HTS18, 19, but conducting a high-throughput (HT)-high-content (HC) screen in neuronal models is problematic due to high cost, environmental variation and human error. In order to detect cellular responses on a 'phenomics' scale using HC imaging one has to reduce variation and error, while increasing sensitivity and reproducibility. 
	Herein we describe a method to accurately and reliably conduct shRNA screens using automated cell culturing20 and HC imaging in neuronal cellular models. We describe how we have used this methodology to identify modulators for one particular protein, DJ1, which when mutated causes autosomal recessive parkinsonism21. 
	Combining the versatility of HC imaging with HT methods, it is possible to accurately quantify a plethora of phenotypes. This could subsequently be utilized to advance our understanding of the genome, the pathways involved in disease pathogenesis as well as identify potential therapeutic targets.

We describe a methodology combining automated cell culturing with high-content imaging to visualize and quantify multiple cellular processes and structures, in a high-throughput manner. Such methods can aid in the further functional annotation of genomes as well as identify disease gene networks and potential drug targets.

在神经退行性疾病的高内涵筛选

The functional annotation of genomes, construction of molecular networks and novel drug target identification, are important challenges that need to be ...

Imaging Glioma Initiation In Vivo Through a Polished and Reinforced Thin-skull Cranial Window

Glioma is the one of the most lethal forms of human cancer. The most effective glioma therapy to date-surgery followed by radiation treatment-offers patients only modest benefits, as most patients do not survive more than five years following diagnosis due to glioma relapse 1,2. The discovery of cancer stem cells in human brain tumors holds promise for having an enormous impact on the development of novel therapeutic strategies for glioma 3. Cancer stem cells are defined by their ability both to self-renew and to differentiate, and are thought to be the only cells in a tumor that have the capacity to initiate new tumors 4. Glioma relapse following radiation therapy is thought to arise from resistance of glioma stem cells (GSCs) to therapy 5-10. In vivo, GSCs are shown to reside in a perivascular niche that is important for maintaining their stem cell-like characteristics 11-14. Central to the organization of the GSC niche are vascular endothelial cells 12. Existing evidence suggests that GSCs and their interaction with the vascular endothelial cells are important for tumor development, and identify GSCs and their interaction with endothelial cells as important therapeutic targets for glioma. The presence of GSCs is determined experimentally by their capability to initiate new tumors upon orthotopic transplantation 15. This is typically achieved by injecting a specific number of GBM cells isolated from human tumors into the brains of severely immuno-deficient mice, or of mouse GBM cells into the brains of congenic host mice. Assays for tumor growth are then performed following sufficient time to allow GSCs among the injected GBM cells to give rise to new tumors-typically several weeks or months. Hence, existing assays do not allow examination of the important pathological process of tumor initiation from single GSCs in vivo. Consequently, essential insights into the specific roles of GSCs and their interaction with the vascular endothelial cells in the early stages of tumor initiation are lacking. Such insights are critical for developing novel therapeutic strategies for glioma, and will have great implications for preventing glioma relapse in patients. Here we have adapted the PoRTS cranial window procedure 16and in vivo two-photon microscopy to allow visualization of tumor initiation from injected GBM cells in the brain of a live mouse. Our technique will pave the way for future efforts to elucidate the key signaling mechanisms between GSCs and vascular endothelial cells during glioma initiation.

Imaging Glioma Initiation In Vivo Through a Polished and Reinforced Thin-skull Cranial Window

By combining a polished and reinforced thin-skull (PoRTS) cranial window and glioblastoma (GBM) cell injection, we can observe glioma initiation and growth from injected GBM cells in the brain of a live mouse longitudinally.

成像神经胶质瘤启动<em在体内</em&gt;通过抛光和增强的薄膜头骨的颅窗

Glioma is the one of the most lethal forms of human cancer. The most effective glioma therapy to date-surgery followed by radiation treatment-offers ...

Voluntary Breath-hold Technique for Reducing Heart Dose in Left Breast Radiotherapy

Breath-holding techniques reduce the amount of radiation received by cardiac structures during tangential-field left breast radiotherapy. With these techniques, patients hold their breath while radiotherapy is delivered, pushing the heart down and away from the radiotherapy field. Despite clear dosimetric benefits, these techniques are not yet in widespread use. One reason for this is that commercially available solutions require specialist equipment, necessitating not only significant capital investment, but often also incurring ongoing costs such as a need for daily disposable mouthpieces. The voluntary breath-hold technique described here does not require any additional specialist equipment. All breath-holding techniques require a surrogate to monitor breath-hold consistency and whether breath-hold is maintained. Voluntary breath-hold uses the distance moved by the anterior and lateral reference marks (tattoos) away from the treatment room lasers in breath-hold to monitor consistency at CT-planning and treatment setup. Light fields are then used to monitor breath-hold consistency prior to and during radiotherapy delivery.

The current priority in breast cancer radiotherapy is to reduce cardiac doses without compromising target tissue coverage. The voluntary breath-hold technique described here is a simple, inexpensive solution to this problem and capable of being instituted widely without the need for specialized equipment.

自愿屏气技术减少心脏剂量的左乳房放射治疗

Breath-holding techniques reduce the amount of radiation received by cardiac structures during tangential-field left breast radiotherapy. With these ...

State of the Art Cranial Ultrasound Imaging in Neonates

Cranial ultrasound (CUS) is a reputable tool for brain imaging in critically ill neonates. It is safe, relatively cheap and easy to use, even when a patient is unstable. In addition it is radiation-free and allows serial imaging. CUS possibilities have steadily expanded. However, in many neonatal intensive care units, these possibilities are not optimally used. We present a comprehensive approach for neonatal CUS, focusing on optimal settings, different probes, multiple acoustic windows and Doppler techniques. This approach is suited for both routine clinical practice and research purposes. In a live demonstration, we show how this technique is performed in the neonatal intensive care unit. Using optimal settings and probes allows for better imaging quality and improves the diagnostic value of CUS in experienced hands. Traditionally, images are obtained through the anterior fontanel. Use of supplemental acoustic windows (lambdoid, mastoid, and lateral fontanels) improves detection of brain injury. Adding Doppler studies allows screening of patency of large intracranial arteries and veins. Flow velocities and indices can be obtained. Doppler CUS offers the possibility of detecting cerebral sinovenous thrombosis at an early stage, creating a window for therapeutic intervention prior to thrombosis-induced tissue damage. Equipment, data storage and safety aspects are also addressed.

Cranial ultrasound (CUS) is a valuable tool for brain imaging in critically ill neonates. This video shows a comprehensive approach for neonatal (Doppler) CUS for both clinical and research purposes, including a bedside demonstration of the technique.

艺术颅超声成像在新生儿的状态

Cranial ultrasound (CUS) is a reputable tool for brain imaging in critically ill neonates. It is safe, relatively cheap and easy to use, even when a ...

A Mouse Model of Orthopedic Surgery to Study Postoperative Cognitive Dysfunction and Tissue Regeneration

Surgery is commonly used to improve and maintain quality of life. Unfortunately, in vulnerable patients such as the elderly, complications may occur and significantly diminish the outcome. Indeed, after routine orthopedic surgery to repair a fracture, as many as 50% of elderly patients suffer from neurologic complications like delirium. Also, the capacity to heal and regenerate tissue after surgery decreases with age, and can impact the quality of fracture repair and even osseous integration of implants. Thus, a better understanding of mechanisms that drive these age-dependent changes could provide strategic targets to minimize risk for such complications and optimize outcomes. Here, we introduce a clinically relevant mouse model of tibial fracture. The postoperative changes in these mice mimic some of the cognitive impairments commonly observed after routine orthopedic surgery in humans. Briefly, an incision is performed in the right hind limb under strictly aseptic conditions. Muscles are disassociated, and a 0.38-mm stainless steel pin is inserted into the upper crest of the tibia, inside the intramedullary canal. Osteotomy is then performed, and the wound is stapled. We have used this model to investigate the effects of surgical trauma on postoperative neuroinflammation and behavioral changes. By applying this fracture model in combination with parabiosis, a surgical model in which 2 mice are anastomosed, we have studied cells and secreted factors that systemically rejuvenate organ function and tissue regeneration after injury. By following our step-by-step protocol, these models can be reproduced with high fidelity, and can be adapted to interrogate many biologic pathways that are altered by surgical trauma.

This protocol describes a mouse model of orthopedic surgery that has been used to study mechanisms of postoperative neuroinflammation and behavioral changes, and when combined with parabiosis, to study tissue regeneration during aging.

骨科手术的小鼠模型研究术后认知功能障碍与组织再生

Surgery is commonly used to improve and maintain quality of life. Unfortunately, in vulnerable patients such as the elderly, complications may occur and ...

Treatment of Liver Metastases Using an Internal Target Volume Method for Stereotactic Body Radiotherapy

The prognosis of patients with metastatic cancers has improved in the past decades due to effective chemotherapy and oligometastatic surgery. For inoperable patients, local ablation therapies, such as stereotactic body radiotherapy (SBRT), can provide effective local tumor control with minimal toxicity. Because of its high precision and accuracy, SBRT delivers a higher radiation dose per fraction, is more effective, and targets smaller irradiation volumes than does conventional radiotherapy. In addition, steep dose gradients from target lesions to surrounding normal tissues are achieved using SBRT; thus, SBRT provides more effective tumor control and exhibits fewer side effects than conventional radiotherapy. The use of SBRT is prevalent for treating intracranial lesions (known as stereotactic radiosurgery); however, it is now also used for treating spinal and adrenal metastases. Because of advancements in image-guided assistance and respiratory motion management, several studies have investigated the use of SBRT for treating lung or liver tumors, which move as a patient breathes. The results of these studies have suggested that SBRT favorably controls tumors in the case of moving lesions.
Four-dimensional computed tomography (4D-CT) with an abdominal compressor (AC) is clinically convenient for effective respiratory motion management. Because this method is noninvasive and allows free breathing, its use reduces complications. Furthermore, patients consider this method convenient. Moreover, it is considered more efficient than other methods of respiratory motion management by physicians and therapists. The use of 4D-CT with an AC for treating pulmonary lesions has also been widely investigated, and the technique is gaining acceptance for treating hepatic lesions. However, the protocols for using 4D-CT with an AC for treating hepatic lesions are different from those used for treating pulmonary lesions. In this article, we describe a new protocol for SBRT with 4D-CT and an AC for treating liver metastases.

Stereotactic body radiotherapy (SBRT) requires rigorous accuracy and precision for delivering high radiation doses per fraction to small treatment volumes to improve tumor control and simultaneously reduce toxicity. Herein, we present a noninvasive and clinically convenient respiratory motion management protocol for SBRT for liver metastases.

内靶容积法治疗肝转移瘤的立体定向体放疗

The prognosis of patients with metastatic cancers has improved in the past decades due to effective chemotherapy and oligometastatic surgery. For ...

Radiation Planning Assistant - A Streamlined, Fully Automated Radiotherapy Treatment Planning System

The Radiation Planning Assistant (RPA) is a system developed for the fully automated creation of radiotherapy treatment plans, including volume-modulated arc therapy (VMAT) plans for patients with head/neck cancer and 4-field box plans for patients with cervical cancer. It is a combination of specially developed in-house software that uses an application programming interface to communicate with a commercial radiotherapy treatment planning system. It also interfaces with a commercial secondary dose verification software. The necessary inputs to the system are a Treatment Plan Order, approved by the radiation oncologist, and a simulation computed tomography (CT) image, approved by the radiographer. The RPA then generates a complete radiotherapy treatment plan. For the cervical cancer treatment plans, no additional user intervention is necessary until the plan is complete. For head/neck treatment plans, after the normal tissue and some of the target structures are automatically delineated on the CT image, the radiation oncologist must review the contours, making edits if necessary. They also delineate the gross tumor volume. The RPA then completes the treatment planning process, creating a VMAT plan. Finally, the completed plan must be reviewed by qualified clinical staff.

Radiation therapy is a highly complex cancer treatment that requires multiple specialists to create a treatment plan and provide quality assurance (QA) prior to delivery to a patient. This protocol describes the use of a fully automated system, the Radiation Planning Assistant (RPA), to create high-quality radiation treatment plans.

放射规划助理-一个流线型的全自动放疗治疗计划系统

The Radiation Planning Assistant (RPA) is a system developed for the fully automated creation of radiotherapy treatment plans, including volume-modulated ...

Assessment of Static Graviceptive Perception in the Roll-Plane using the Subjective Visual Vertical Paradigm

Vestibular disorders are among the most common syndromes in medicine. In recent years, new vestibular diagnostic systems have been introduced that allow the examination of all semicircular canals in the clinical setting. Assessment methods of the otolithic system, which is responsible for the perception of linear acceleration and perception of gravity, are far less in clinical use. There are several experimental approaches for measuring the perception of gravity. The most frequently used method is the determination of the subjective visual vertical. This is usually measured with the head in an upright position. We present here an assessment method for testing otolith function in the roll plane. The subjective visual vertical is measured in the head upright position as well as with head inclination of &#177; 15&#176; and &#177; 30&#176; in the roll plane. This extended functional paradigm is an easy-to-perform clinical test of otolith function and ensures increased information content for the detection of impaired graviceptive perception.

The perception of gravity is commonly determined by the subjective visual vertical in the head upright position. The additional assessment at head tilts of &#177; 15&#176; and &#177; 30&#176; in the roll plane ensures increased information content for the detection of impaired graviceptive perception.

使用主观视觉垂直范式对卷平面中的静态视觉感知的评估

Vestibular disorders are among the most common syndromes in medicine. In recent years, new vestibular diagnostic systems have been introduced that allow ...