基廷博士是由美国国防部资助CA100335资助，是圣Baldrick基金会学者。

所有上述程序进行审查和批准我们的机构动物照顾及使用委员会。 1，规划实验<ol><li>选择的细胞被注入。从各种来源的细胞是候选注射：贴壁细胞培养系，遗传修饰的克隆，神经干细胞，原代培养物，或分类肿瘤。模型所需的类型将确定注射的最适当的站点。 <ol><li>确定细胞数的注射。形成肿瘤所需的细胞数量与细胞系而不同，必须根据经验确定;肿瘤生长速率很大程度上依赖于细胞类型，细胞数目和组织培养传代次数。细胞数为1×10 4〜2×10 5以上每次注射都产生了不同的生长率的肿瘤。 </li><li>限制用于注射的体积。细胞应在无血清培养基或磷酸盐缓冲S的体积最小暂停艾琳（PBS），允许通过＆G针光滑，不易通过。较小的体积实际上输送到大脑，更精确的和所限定的所得肿瘤;最佳的结果是通过注入体积为3至6微升获得。打算制备至少50微升细胞悬浮液的最终的总体积，而不论计划注射的次数的，允许精确测量，混合和采样。 </li></ol></li><li>选择合适的老鼠模型。人类肿瘤的小鼠模型中通常使用年轻免疫妥协小鼠以避免免疫介导的移植物排斥。这是没有必要的工作，以发生在生物安全柜中，如果​​适当的预防措施，包括去污，消毒，以及对材料和设备灭菌。这里示出的工作已在无胸腺裸鼠，雄性和雌性已经完成，6周和12周的年龄之间。该称重小于20g可以是困难的小鼠到位置上进行tereotaxic帧，并且可以更容易受到低温。 </li></ol> 2，装配设备<ol><li>获取并净化鼠标立体框架，调整控制台，鼠标气体麻醉头部固定器，微量注射泵，热板机，麻醉机，光纤工作灯，和变速回转钻机。清洁和消毒所有设备。 </li><li>编程的注射参数（包括注射体积，流速，速度的单位，注射器型）和其他变量所需的注射器泵的操作。优化设置，为每个特定的模式。 注意：在这些注射的设置分别为3000 NL样品在400升/分钟的速度（速度单位“M”）用25微升注射器（设备类型“E”），在非分组（“N”）模式。 </li><li>清洁精密微量与＆G针头无菌去离子水冲洗几次（DIH 2 O）和70％的乙醇（乙醇），做最后的RINSE与DIH 2 O。一些但不是所有的模型被设计成可高压灭菌。确保柱塞运动是平滑和自由。 </li><li>获得麻醉交运设备。异氟醚是优选的麻醉剂，因为它很容易调节麻醉深度和持续时间。确保该立体定位单元配备一个鼠标麻醉气体附着和必要的管道和连接器的位置，从麻醉机提供的气体混合物的小鼠。 </li><li>高压釜中的外科手术工具，包括精尖的剪刀，二镊子/止血剂（有齿），用于缝合，中等大小的剪刀，中期和细尖镊子，以及至少两个1毫米牙科钻头。用70％乙醇，消毒液，或珠灭菌器在操作过程中保持仪器的清洁。 </li><li>从获得的异氟烷，镇痛药，眼用软膏/润滑剂，抗生素软膏，盐水，30％过氧化氢 ​​（H 2 O 2），防腐剂，和骨蜡（如果需要的话）兽医和医疗用品。准备无菌DIH 2 O和70％的乙醇。一次性用品如纱布，无菌敷料，缝合线，乙醇棉签，棉签拭子，无菌手术手套，手术刀，更是详细列出材料清单。的细尖永久性标记用于标记头骨应进行消毒，并专门用于外科手术中使用。 </li></ol> 3，准备细胞注射<ol><li>在这里所示的实验中，选择一个人永生化的GBM细胞系。确定细胞的数量（2×10 5）和悬浮液的体积被注入（3微升）经验;细节可以与细胞类型而有所不同。使用至少50微升的体积，方便精确的测量，混合和注射。 </li><li> Trypsinize细胞重悬在媒体或PBS。执行以下步骤之前，以细胞注射： <ol><li>通过抽吸去除细胞介质和冲洗用10毫升的PBS，每100毫米的钢板;除去PBS。 </li><li>广告d 1的ml胰蛋白酶以每次100毫米的钢板并在室温下孵育足够长的时间以得到单细胞悬浮液。 </li><li>停止胰蛋白酶的活性用5毫升贝科的改良Eagle培养基（DMEM）+10％胎牛血清（FBS）每100毫米的钢板。 </li><li>离心沉淀细胞在450 XG重悬在每100毫米的钢板〜5ml无血清的DMEM（SF-DMEM）。 </li><li>计数使用血球计数器或自动细胞计数活细胞。 </li><li>调节活细胞密度为2×10 5个细胞/ 3微升（或通过实验测定）通过制粒的细胞和再悬浮在SF-DMEM。 </li></ol></li><li>保持细胞冷冻在冰或冷包（不要让细胞冻结）。通过手指轻弹，轻轻混匀。 </li></ol> 4，麻醉并准备鼠标外科手术<ol><li>诱导麻醉用〜5％的异氟醚（氧流量〜2升/分钟）或按照兽医。电磁炉应取2〜3分钟。鼠标保持在适当的深度的麻醉〜3％的异氟烷​​。 <ol><li>检查麻醉深度脚趾捏，如果显示调整异氟烷，并持续监控整个手术过程中的呼吸和脚趾捏响应。覆盖鼠标，用纱布垫保暖并在整个过程中，这可能需要45分钟到1小时，这取决于注入的速率定期监测小鼠。 </li></ol></li><li>通过仔细地滑口感棒在舌头和放入口中放置鼠标在立体框架。挂钩的前门牙到牙齿洞。使用耳棒以稳定头部，与耳朵到眼睛表面尽量接近水平越好。不要强行进入酒吧耳道。关键是要牢固地定位头：轻轻检查一侧到另一侧与向上和向下运动，用指尖。利用立体定位装置的调节控制，优化配合。 </li><li>润滑眼睛眼药膏。清洁耳朵和眼睛之间的皮肤用聚维酮碘比交替应用的两倍Ë防腐剂和70％的乙醇。 </li><li>选择注射止痛皮下（SQ）为沿（或遵医嘱）。 </li></ol> 5，注射执行<ol><li>确定注射部位。注射部位可在大小和小鼠品系与细胞的类型而变化。细胞注射太靠近心室可能导致疾病的颅脊椎蔓延。细胞注射太浅，可能会生长出通过针轨道。 请注意：在这里所示的实验中，目标被定义为大脑皮质的额叶区域的站点2.5毫米横向（右），1.5毫米前壁，和3.5毫米腹侧相对于前囟点被选择（ 图1），产生中脑肿瘤的小鼠，从20至30g。 </li><li>使用无菌技术通过头部的皮肤，以暴露前囟和注射部位的一小切口（〜长8毫米）。的斜切口，从中线轴线并朝向右耳右眼（之间的点<strong&gt;图2）允许将前囟门和注射部位。 </li><li>牵开皮肤，用无菌棉签擦干头骨表面。吸干骨与另一个棉签蘸有H 2 O 2形象化前囟门。的H 2 O 2将产生氧气气泡，而使细线的白色泡沫状物沿相交于前囟的冠状面和矢状缝。 </li><li>锁针空的注射器上的微型泵和机动针直接在囟门的顶端;对准控制台上设置的坐标为0.0毫米横向，0.0毫米前/后，以及0.0毫米腹/背。 </li><li>移动针至2.5mm的横向（右）至1.5mm前壁相对于前囟（或所希望的位置）用的控制旋钮的立体定位装置上。提高注射器略有并标记上配有专用毡尖笔头骨的确切位置。如果头骨的外表面不与前囟门（I A级平面。即，在背/腹坐标不再读数为零），复位腹侧/背侧读取到0.0，以确保注射的深度不偏移。 </li><li>钻一小孔与手持式回转钻机配备在标有步骤5.5钢笔相应部位的无菌牙尖的头骨。保持钻头以一个角度向颅骨和非常轻微接触尖端到骨。根据需要重复。钻几乎但不完全穿过骨，留针以实际穿透该层的头骨，结果在最干净的注射。 </li><li>去除骨扬灰，用棉签蘸在PBS或生理盐水。回到注射器泵和降低针直线下降到钻孔中，以确认位置。如果钻孔不居中的针，然后用电钻扩大开放。 </li><li>轻轻混合细胞悬液和绘制的细胞分化成使用泵控制器的注射器。避免气泡和团块，然后擦拭针头机智ħ酒精棉签去除的外表面污染的细胞。如果注射器/针头堵塞或冷冻，取出，并迅速干净，用无菌水和70％乙醇。 </li><li>降低针的颅骨表面的水平（0.0毫米腹/背部）。然后慢慢地（经过大约1分钟）降低针头刺穿颅骨的整个厚度和穿透脑向4毫米ventrall的深度。慢慢地拔出针头至3.5mm腹侧。如果鼠标头在立体部牢固地保持，应该有颅骨几乎没有移动。 </li><li>确认正确的参数输入到泵控制器（2.2节），然后按“运行/停止”（或按照产品说明书），以autoinject细胞。监视鼠标和指示，如果调整​​的异氟醚。观看的注射器，并确保该柱塞被朝着桶​​。冷冻或堵塞移动可导致弯曲/断柱塞。 </li><li>等待1到2分钟，在B的针雨，然后非常缓慢地（超过3至4分钟）从组织收回针头。总时间以除去针注射后是完整的是5分钟。用棉签吸干周围钻孔的面积;离开皮肤开的边缘，以允许所述骨干。 </li><li>从泵中取出注射器和快速冲洗3次，用无菌H 2 O，3次用70％乙醇和3倍，用无菌水（或按照制造商）。擦拭针头乙醇，备用。 </li><li>应用无菌骨蜡（相当于1或2微升）到所述钻孔并使用无菌棉签的木端夯实蜡上并进入骨骼。如果表面被充分地干燥，则应该坚持。 </li><li>散布无菌覆盖在鼠标周围的工作区和缝合切口;三针应该是足够的。或者，可使用外科手术粘合剂。涂抹外用抗生素。 </li><li>异氟醚降低至0％，并从立体定位装置移开鼠标，是Careful保护牙齿。检查耳朵，嘴巴和舌头。 </li><li>监控鼠标密切关注注射。在5〜10分钟的热垫鼠标的地方;然后转移到笼子里（上隔热垫），并观察到鼠标的唤醒，是门诊。不要让鼠标无人值守，直到它重新获得足够的意识来维持胸骨recumbence。不要返回鼠标其他动物的公司，直到完全康复。考虑使用额外的镇痛如果疼痛的迹象注意到。 </li></ol> 6，完成并监控恢复与肿瘤发展<ol><li>清洁所有采用70％乙醇，老鼠之间的珠灭菌或消毒仪器和设备。 </li><li>对于疼痛，感染或其他并发症的征兆的步骤后监测小鼠注射两天。注入镇痛在24和48小时（或指示）过程之后。如果有必要在7〜10天取出缝线。 </li><li>评估老鼠的疾病，paralysi临床症状秒，活性降低，体重减轻，癫痫发作，或一般疾病。 </li><li>监测肿瘤发展通过适当的方法[磁共振成像（MRI）， 体内成像系统（IVIS）等]。 </li></ol>

<ol>
	<li>Heyer, J., Kwong, L. N., Lowe, S. W., Chin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-germline+genetically+engineered+mouse+models+for+translational+cancer+research.">Non-germline genetically engineered mouse models for translational cancer research.</a> Nature reviews. Cancer. 10, 470-480 (2010).</li><li>Yamada, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+to+accurately+inject+tumor+cells+into+the+caudate/putamen+nuclei+of+the+mouse+brain.">A method to accurately inject tumor cells into the caudate/putamen nuclei of the mouse brain.</a> The Tokai journal of experimental and clinical medicine. 29, 167-173 (2004).</li><li>Brooks, A. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproducible+and+efficient+murine+CNS+gene+delivery+using+a+microprocessor-controlled+injector.">Reproducible and efficient murine CNS gene delivery using a microprocessor-controlled injector.</a> Journal of neuroscience. 80, 137-147 (1998).</li><li>Shankavaram, U. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+profiling+indicates+orthotopic+xenograft+of+glioma+cell+lines+simulate+a+subclass+of+human+glioblastoma.">Molecular profiling indicates orthotopic xenograft of glioma cell lines simulate a subclass of human glioblastoma.</a> Journal of cellular and molecular medicine. 16, 545-554 (2012).</li><li>Giannini, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient+tumor+EGFR+and+PDGFRA+gene+amplifications+retained+in+an+invasive+intracranial+xenograft+model+of+glioblastoma+multiforme.">Patient tumor EGFR and PDGFRA gene amplifications retained in an invasive intracranial xenograft model of glioblastoma multiforme.</a> Neuro-oncology. 7, 164-176 (2005).</li><li>Yi, D., Hua, T. X., Lin, H. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EGFR+gene+overexpression+retained+in+an+invasive+xenograft+model+by+solid+orthotopic+transplantation+of+human+glioblastoma+multiforme+into+nude+mice.">EGFR gene overexpression retained in an invasive xenograft model by solid orthotopic transplantation of human glioblastoma multiforme into nude mice.</a> Cancer investigation. 29, 229-239 (2011).</li><li>Carty, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracranial+injection+of+AAV+expressing+NEP+but+not+IDE+reduces+amyloid+pathology+in+APP+PS1+transgenic+mice.">Intracranial injection of AAV expressing NEP but not IDE reduces amyloid pathology in APP+PS1 transgenic mice.</a> PLos ONE. 8, e59626 (2013).</li><li>Thaci, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacokinetic+study+of+neural+stem+cell-based+cell+carrier+for+oncolytic+virotherapy:+targeted+delivery+of+the+therapeutic+payload+in+an+orthotopic+brain+tumor+model.">Pharmacokinetic study of neural stem cell-based cell carrier for oncolytic virotherapy: targeted delivery of the therapeutic payload in an orthotopic brain tumor model.</a> Cancer gene therapy. 19, 431-442 (2012).</li><li>Ozawa, T., 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=Establishing+intracranial+brain+tumor+xenografts+with+subsequent+analysis+of+tumor+growth+and+response+to+therapy+using+bioluminescence+imaging.">Establishing intracranial brain tumor xenografts with subsequent analysis of tumor growth and response to therapy using bioluminescence imaging.</a> J. Vis. Exp. (41), (2010).</li><li>Valadez, J. G., Sarangi, A., Lundberg, C. J., Cooper, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+orthotopic+glioma+xenografts+recapitulate+infiltrative+growth+and+isocitrate+dehydrogenase+I+mutation.">Primary orthotopic glioma xenografts recapitulate infiltrative growth and isocitrate dehydrogenase I mutation.</a> J. Vis. Exp. (83), (2014).</li><li>Baumann, B. C., Dorsey, J. F., Benci, J. L., Joh, D. Y., Kao, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereotactic+intracranial+implantation+and+in+vivo+bioluminescent+imaging+of+tumor+xenografts+in+a+mouse+model+system+of+glioblastoma+multiforme.">Stereotactic intracranial implantation and in vivo bioluminescent imaging of tumor xenografts in a mouse model system of glioblastoma multiforme.</a> J. Vis. Exp. (67), (2012).</li><li>Iwami, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+of+intracranial+injection+via+the+postglenoid+foramen+for+brain+tumor+mouse+models.">A novel method of intracranial injection via the postglenoid foramen for brain tumor mouse models.</a> Journal of neurosurgery. 116, 630-635 (2012).</li></ol>

作者什么都没有透露。

人脑肿瘤的原位小鼠模型可以是一个很好的工具，用于评估临床治疗的效果，但必须小心，以优化布局细胞的脑组织中。研究表明，过量的等分试样体积，次优的喷射技术和匆忙的注射速率可导致泄漏和肿瘤细胞中不希望的位置（脑室，脊髓硬膜外区域等 ）的外观和高变异在肿瘤大小为2（个人观察）。非细胞样品的微处理器驱动输送的分析发现，使用的微型泵的制造更focalized交货...

在脑肿瘤细胞的体外研究是非常宝贵的解剖驾驶生长，存活，迁移和侵袭癌细胞的分子机制;培养细胞实验中可以定义信号传导通路，提出潜在的治疗靶点，并描述对药物治疗的细胞反应。但在体外系统过于简单化预测到药品有机体反应;他们缺乏的生理反应，免疫反应，细胞微环境和活生生的动物系统的整体异质性。基因工程模型可以是无价的，当可用的，但不同的分子种类和鼠细胞之间存在着可能无法概括人类进程的事件，造成显著差异比较动物模型的临床观察1时。鼠标涉及皮下（SQ）注射人脑肿瘤细胞系的侧翼的皮肤下异种移植模型中很容易就可以执行和衡量;它们可以被用来处理基因修饰和药物施用/输送，代谢和毒性的影响。显著缺点，但是，限制SQ模型的实用性。微环境并没有概括了一个自然发生​​的脑肿瘤：不同类型的细胞和组织的相互作用;局部血管，以及无数的其他因素所特有的大脑是无法复制的。为了更准确地再现了自然发生脑肿瘤的独特氛围和测试药物干预的效果，鼠标原位模型应该加以利用。此外，原位技术可以被用来作为一种基因工程的方法，其中人类初级的非癌性细胞（分化的细胞或祖）的一部分被遗传修饰，并注射到小鼠的相关部位，具有或不具有人的基质细胞，导致肿瘤发生在人中1所见相似的。 本文介绍一种方法来精确和可重复产生脑肿瘤的小鼠。使用该技术，用户可以准确地注入少量等份悬浮细胞到小鼠大脑皮质的额叶后部顶颞区的指定位置。鼠标死亡率极低;在我们的手中，没有小鼠的后185程序从手术并发症死亡。将所得的肿瘤的特征可以与典型的人类临床肿瘤进行比较;例如：生长，坏死的程度，浸润程度的细胞类型，有丝分裂细胞的存在下，细胞增殖和细胞凋亡等的标志物的异质性快速性的细胞系或分类的人体组织或肿瘤样品随后可以根据自己的能力进行评价以模拟实际的临床表现。医药品，选定基于它们在细胞培养物的性能，可以在一个正常运作代谢，循环系统，和血 - 脑屏障的情况下进行测试，因为它们在动物背负机智存在医管局肿瘤，在所有相关的建筑环境。此外，选择用于注射的细胞可以被遗传修饰以探讨特定击倒，缺失的影响，敲插件，突变等，对肿瘤生长和存活。 许多出版物文档中使用的各种颅内肿瘤的技术研究。 Yamada 等人做了注射U87细胞的染料和进行详细研究，结果发现最小体积和喷射率产生最佳的肿瘤2。 布鲁克斯等人发现使用微处理器控制的喷油器，而不是人工的方法来实现病毒载体卓越的重现性和效率;他们对最佳注射参数的结论也适用于小区交付3。 Shankavaram 等人发现，胶质母细胞瘤（GBM）细胞系注射原位（使用手动方法）进入大脑概括了CL的基因表达谱inical肿瘤更加紧密地比在体外或倚移植，配套使用颅内模型的临床前研究4。尼尼等注入从已持续在裸鼠 ​​的侧腹通过连续传代至另外的小鼠的脑中人类手术标本的细胞，并表明，这种方法保藏患者肿瘤基因改变模型中的5。类似的结果，报告毅等[6]。采用立体设置，仔细界定注射部位，并缓慢而稳定的注入量，他们获得可重复的脑瘤一致的增长率和高（100％）植入率。此技术的有效性也因此被确立;文献检索表明，这一技术的应用非常广泛。卡蒂等人使用颅内注射成功提供病毒载体表达治疗基因进入transgeni的额叶皮质阿尔茨海默氏病7的C型号。特哈契等人描述了使用颅内注射递送治疗的溶瘤腺病毒在神经干细胞的基础载体为已携带原位注射的GBM肿瘤8裸鼠中。显然，颅内注射是一种多用途的和有效的工具，临床前研究。在可视化实验杂志此前出版物描述的基本方法9-11，但我们采用易于掌握技术把颅内肿瘤注射原位模型的概念，精度更高的水平。

<table border="0" cellpadding="0" cellspacing="0" width="866">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Name of Reagent/ Equipment</td>
			<td style="width:87px;">Company</td>
			<td style="width:145px;">Catalog Number</td>
			<td style="width:287px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Equipment</td>
			<td style="width:87px;"></td>
			<td style="width:145px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:349px;">Small Animal Stereotaxic Instrument with Digital Display Console.</td>
			<td style="width:87px;">Kopf</td>
			<td style="width:145px;">Model 940</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Mouse Gas Anesthesia Head Holder</td>
			<td style="width:87px;">Kopf</td>
			<td style="width:145px;">Model 923-B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Mouse Ear Bars</td>
			<td style="width:87px;">Kopf</td>
			<td style="width:145px;">Medel 922</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Fiber Optic Illuminator</td>
			<td style="width:87px;">Fisher</td>
			<td style="width:145px;">12-562-36</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">UltraMicroPump III</td>
			<td style="width:87px;">WPI</td>
			<td style="width:145px;">UMP3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Micro4 microprocessor</td>
			<td style="width:87px;">WPI</td>
			<td style="width:145px;">UMC4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Variable speed hand-held rotary drill</td>
			<td style="width:87px;">Dremel</td>
			<td style="width:145px;">Model 300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Dental drill bit, 1.0 mm</td>
			<td style="width:87px;">Spoelting</td>
			<td style="width:145px;">514554</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Adaptor for dental drill bit: 3/32 inch collet</td>
			<td style="width:87px;">Dremel</td>
			<td style="width:145px;">481</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Heating pad</td>
			<td style="width:87px;"></td>
			<td style="width:145px;"></td>
			<td>for mice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Isoflurane vaporizer system</td>
			<td style="width:87px;"></td>
			<td style="width:145px;"></td>
			<td>for mice</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:349px;">Medical tubing and connectors</td>
			<td style="width:87px;"></td>
			<td style="width:145px;"></td>
			<td style="width:287px;">to connect isoflurane vaporizer with stereotaxic frame</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Instruments</td>
			<td style="width:87px;"></td>
			<td style="width:145px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Precision 25 ul micro syringe</td>
			<td style="width:87px;">Hamilton&#160;</td>
			<td style="width:145px;">7636-01</td>
			<td>Model 702, without needle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Microsyringe needles, 26s gauge&#160;</td>
			<td style="width:87px;">Hamilton&#160;</td>
			<td style="width:145px;">7804-04</td>
			<td>RN, 25 mm point style 2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Fine-tipped scissors (straight, sharp/sharp)</td>
			<td style="width:87px;"></td>
			<td style="width:145px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Medium-sized standard scissors</td>
			<td style="width:87px;"></td>
			<td style="width:145px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Standard serrated forceps</td>
			<td style="width:87px;"></td>
			<td style="width:145px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Serrated hemostats (2)</td>
			<td style="width:87px;"></td>
			<td style="width:145px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Fine-tipped forceps</td>
			<td style="width:87px;"></td>
			<td style="width:145px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Supplies</td>
			<td style="width:87px;"></td>
			<td style="width:145px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Sutures 5-0 vicryl P-3 13 mm (Ethicon)</td>
			<td style="width:87px;">MWI</td>
			<td style="width:145px;">J463G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Surgical blades #10, stainless (Feather)</td>
			<td style="width:87px;">Fisher</td>
			<td style="width:145px;">296#10</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:349px;">Isoflurane (Fluriso)&#160;</td>
			<td style="width:87px;">VetOne&#160;</td>
			<td style="width:145px;">NDC 13985-528-60</td>
			<td style="width:287px;">Item #502017. Liquid inhalation anesthetic. federal law restricts this drug to use by or on the order of a licensed veterinarian.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Carprofen (Rimadyl Injectable 50 mg/mL)&#160;</td>
			<td style="width:87px;">Pfizer</td>
			<td style="width:145px;">NDC 61106-8507-01</td>
			<td>dilute in saline</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Ophthalmic ointment (artificial tears)</td>
			<td style="width:87px;">Rugby</td>
			<td style="width:145px;">NDC 0536-6550-91</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Topical antibiotic (AK-Poly-Bac )</td>
			<td style="width:87px;">Akorn</td>
			<td style="width:145px;">NDC 17478-238-35</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Povidone-iodine topical antiseptic, 10% (Betadine)</td>
			<td style="width:87px;">Betadine</td>
			<td style="width:145px;">NDC 67618-150-04</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Hydrogen Peroxide, 30%</td>
			<td style="width:87px;">Fisher&#160;</td>
			<td style="width:145px;">H325-100</td>
			<td>for visualizing skull landmarks</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Sterile saline</td>
			<td style="width:87px;">VetOne&#160;&#160;</td>
			<td style="width:145px;">NDC 13985-807-25</td>
			<td>for diluting solutions, cleaning tissue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Bone wax</td>
			<td style="width:87px;">WPI</td>
			<td style="width:145px;">Item #501771</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Sterile drapes</td>
			<td style="width:87px;">McKesson</td>
			<td style="width:145px;">25-517</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Sterile surgical gloves</td>
			<td style="width:87px;">McKesson</td>
			<td style="width:145px;">(to fit)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Sterile gauze pads, 2 x 2</td>
			<td style="width:87px;">Fisherbrand&#160;</td>
			<td style="width:145px;">22028556</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Sterile gauze pads, 4 x 4</td>
			<td style="width:87px;">Fisherbrand&#160;</td>
			<td style="width:145px;">22-415-469</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Alcohol prep pads (medium)</td>
			<td style="width:87px;">PDI</td>
			<td style="width:145px;">B603</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Sterile cotton-tipped applicators</td>
			<td style="width:87px;">Fisherbrand&#160;</td>
			<td style="width:145px;">23-400-114</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:349px;">Sterile 0.5 ml screw cap tube with caps for cells</td>
			<td style="width:87px;">USA Scientific</td>
			<td style="width:145px;">1405-4700</td>
			<td>for cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Individually wrapped sterile dispo pipettes</td>
			<td style="width:87px;">Fisher</td>
			<td style="width:145px;">BD 357575</td>
			<td>for needle cleaning solutions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">BD insulin syringes with needles&#160;</td>
			<td style="width:87px;">Fisher</td>
			<td style="width:145px;">329461</td>
			<td>for analgesic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">70% ethanol</td>
			<td style="width:87px;"></td>
			<td style="width:145px;"></td>
			<td>for cleaning</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:349px;">Sterile di H2O</td>
			<td style="width:87px;"></td>
			<td style="width:145px;"></td>
			<td>for cleaning</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Microfuge tubes for cleaning solutions</td>
			<td style="width:87px;"></td>
			<td style="width:145px;"></td>
			<td>for needle cleaning solutions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Felt tip pen (dedicated)</td>
			<td style="width:87px;"></td>
			<td style="width:145px;"></td>
			<td>for marking skull</td>
		</tr>
	</tbody>
</table>

creating anatomically accurate and reproducible intracranial xenografts of human brain tumors

原位肿瘤模型目前正在研究一种肿瘤类型的特点，使用和不干预的最佳方式，在一个活的动物的情况下-尤其是在遗址具有独特的生理和建筑品质，如大脑在体外和异位模型不能。考虑功能，如血管，血脑屏障，代谢，药物输送和毒性，以及一系列其他相关因素。原位模型有其局限性太多，但与感兴趣适当技术的肿瘤细胞能够正确地植入到组织，在人类大脑最密切模仿的条件。通过采用能够提供精确测量的量在一个一致的速率和压力下，人脑肿瘤具有可预测的增长率的小鼠模型可再现地创建准确的限定位置，并适合各种干预的可靠的分析方法。这里描述的方案着重于德设计和准备的颅内注射，进行了手术，并确保肿瘤的生长成功的和可重复的技术注释细节并提供出发点的各种情况，可定制各种不同的脑肿瘤模型。

可靠的颅内异种移植物可在此描述的技术来创建。识别小鼠头盖骨的关键结构（ 图1）将允许对识别的前囟和引导研究者以一个精确的和可重复的注射位置。在这些研究中的U251亲本系，转染的荧光素酶（U251-吕克）或U87 U251细胞永生化人GBM组织培养细胞在4至6微升SF-DMEM中悬浮，并注射2.5毫米横向（右），1.5毫米前，和3.5毫米腹侧相对于前囟点（ 图2）。 <p class="jove_content...

Watch this Scientific Journal Video about Creating Anatomically Accurate and Reproducible Intracranial Xenografts of Human Brain Tumors at JoVE.com

Creating Anatomically Accurate and Reproducible Intracranial Xenografts of Human Brain Tumors

大脑与那些没有得到很好的利用体外或异位的分析代表素质唯一的站点。原位小鼠模型重现的位置及生长特点可以用立体定位仪固定和低压注射泵颅内注射可靠地创建。

创建精确的解剖学和人脑肿瘤的重现性颅内移植瘤

Orthotopic tumor models are currently the best way to study the characteristics of a tumor type, with and without intervention, in the context of a live ...

creating-anatomically-accurate-reproducible-intracranial-xenografts

Research

JoVE Journal

Medicine

15.4K 次观看.  University of Colorado School of Medicine. 大脑与那些没有得到很好的利用体外或异位的分析代表素质唯一的站点。原位小鼠模型重现的位置及生长特点可以用立体定位仪固定和低压注射泵颅内注射可靠地创建。 

视频: Creating Anatomically Accurate and Reproducible Intracranial Xenografts of Human Brain Tumors

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

在免疫缺陷小鼠的建立和传播人类视网膜母细胞瘤肿瘤

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

科研

医学

Automated Midline Shift and Intracranial Pressure Estimation based on Brain CT Images

In this paper we present an automated system based mainly on the computed tomography (CT) images consisting of two main components: the midline shift estimation and intracranial pressure (ICP) pre-screening system. To estimate the midline shift, first an estimation of the ideal midline is performed based on the symmetry of the skull and anatomical features in the brain CT scan. Then, segmentation of the ventricles from the CT scan is performed and used as a guide for the identification of the actual midline through shape matching. These processes mimic the measuring process by physicians and have shown promising results in the evaluation. In the second component, more features are extracted related to ICP, such as the texture information, blood amount from CT scans and other recorded features, such as age, injury severity score to estimate the ICP are also incorporated. Machine learning techniques including feature selection and classification, such as Support Vector Machines (SVMs), are employed to build the prediction model using RapidMiner. The evaluation of the prediction shows potential usefulness of the model. The estimated ideal midline shift and predicted ICP levels may be used as a fast pre-screening step for physicians to make decisions, so as to recommend for or against invasive ICP monitoring.

An automated midline shift estimation and intracranial pressure (ICP) pre-screening system based on computed tomography (CT) images for patients with traumatic brain injury (TBI) is proposed using image processing and machine learning techniques.

根据脑CT图像的自动中线移位和颅内压的估计

In this paper we present an automated system based mainly on the computed tomography (CT) images consisting of two main components: the midline shift ...

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality treatment consisting of surgical resection, post-operative radiation therapy and temozolomide chemotherapy 1. New therapeutic approaches are clearly needed to improve patient survival and quality of life. The development of more effective treatment strategies would be aided by animal models of GBM that recapitulate human disease yet allow serial imaging to monitor tumor growth and treatment response. In this paper, we describe our technique for the precise stereotactic implantation of bio-imageable GBM cancer cells into the brains of nude mice resulting in tumor xenografts that recapitulate key clinical features of GBM 2. This method yields tumors that are reproducible and are located in precise anatomic locations while allowing in vivo bioluminescent imaging to serially monitor intracranial xenograft growth and response to treatments 3-5. This method is also well-tolerated by the animals with low perioperative morbidity and mortality.

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

We describe an integrated method for the precise, stereotactic implantation of human glioblastoma multiforme cells into the brains of nude mice and subsequent serial in vivo imaging to monitor growth and response to treatment of the resultant xenografts.

立体定向颅内植入和<em在体内</em生物发光成像的肿瘤异种移植的小鼠模型系统的多形性胶质母细胞瘤

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

一种新型的高分辨率<em&gt;在体内</em&gt;成像技术研究肿瘤生长及治疗颅内结构的动态响应

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

In vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate the human disease provide a valuable platform for research into disease pathophysiology and for the preclinical evaluation of novel therapies. We present a variety of methods to generate subcutaneous or orthotopic human HCC xenografts in immunodeficient mice that could be utilized in a variety of research applications. With a focus on the use of primary tumor tissue from patients undergoing surgical resection as a starting point, we describe the preparation of cell suspensions or tumor fragments for xenografting. We describe specific techniques to xenograft these tissues i) subcutaneously; or ii) intrahepatically, either by direct implantation of tumor cells or fragments into the liver, or indirectly by injection of cells into the mouse spleen. We also describe the use of partial resection of the native mouse liver at the time of xenografting as a strategy to induce a state of active liver regeneration in the recipient mouse that may facilitate the intrahepatic engraftment of primary human tumor cells. The expected results of these techniques are illustrated. The protocols described have been validated using primary human HCC samples and xenografts, which typically perform less robustly than the well-established human HCC cell lines that are widely used and frequently cited in the literature. In comparison with cell lines, we discuss factors which may contribute to the relatively low chance of primary HCC engraftment in xenotransplantation models and comment on technical issues that may influence the kinetics of xenograft growth. We also suggest methods that should be applied to ensure that xenografts obtained accurately resemble parent HCC tissues.

Human tumor xenografts in immunodeficient mice are valuable tools to study cancer biology. Specific protocols to generate subcutaneous and intrahepatic xenografts from human hepatocellular carcinoma cells or tumor fragments are described. Liver regeneration induced by partial hepatectomy in recipient mice is presented as a strategy to facilitate intrahepatic engraftment.

皮下及肝内人肝癌裸鼠移植瘤的免疫缺陷小鼠的产生

In  vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate  the human disease provide a valuable platform for research into disease  ...

Lesion Explorer: A Video-guided, Standardized Protocol for Accurate and Reliable MRI-derived Volumetrics in Alzheimer's Disease and Normal Elderly

Obtaining in vivo human brain tissue volumetrics from MRI is often complicated by various technical and biological issues. These challenges are exacerbated when significant brain atrophy and age-related white matter changes (e.g. Leukoaraiosis) are present. Lesion Explorer (LE) is an accurate and reliable neuroimaging pipeline specifically developed to address such issues commonly observed on MRI of Alzheimer's disease and normal elderly. The pipeline is a complex set of semi-automatic procedures which has been previously validated in a series of internal and external reliability tests1,2. However, LE's accuracy and reliability is highly dependent on properly trained manual operators to execute commands, identify distinct anatomical landmarks, and manually edit/verify various computer-generated segmentation outputs.
LE can be divided into 3 main components, each requiring a set of commands and manual operations: 1) Brain-Sizer, 2) SABRE, and 3) Lesion-Seg. Brain-Sizer's manual operations involve editing of the automatic skull-stripped total intracranial vault (TIV) extraction mask, designation of ventricular cerebrospinal fluid (vCSF), and removal of subtentorial structures. The SABRE component requires checking of image alignment along the anterior and posterior commissure (ACPC) plane, and identification of several anatomical landmarks required for regional parcellation. Finally, the Lesion-Seg component involves manual checking of the automatic lesion segmentation of subcortical hyperintensities (SH) for false positive errors.
While on-site training of the LE pipeline is preferable, readily available visual teaching tools with interactive training images are a viable alternative. Developed to ensure a high degree of accuracy and reliability, the following is a step-by-step, video-guided, standardized protocol for LE's manual procedures.

Lesion Explorer: A Video-guided, Standardized Protocol for Accurate and Reliable MRI-derived Volumetrics in Alzheimer&#039;s Disease and Normal Elderly

Lesion Explorer (LE) is a semi-automatic, image-processing pipeline developed to obtain regional brain tissue and subcortical hyperintensity lesion volumetrics from structural MRI of Alzheimer's disease and normal elderly. To ensure a high level of accuracy and reliability, the following is a video-guided, standardized protocol for LE's manual procedures.

病变资源管理器：一个视频引导，标准化为老年痴呆症准确，可靠的MRI衍生Volumetrics和正常老年人协议

Obtaining in vivo human brain tissue volumetrics from MRI is often complicated by various technical and biological issues. These challenges are ...

Minimally Invasive Establishment of Murine Orthotopic Bladder Xenografts

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable cell lines are inoculated either by intravesical instillation (model of nonmuscle invasive growth) or intramural injection into the bladder wall (model of invasive growth). Both procedures are complex and highly time-consuming. Additionally, the superficial model has its shortcomings due to the lack of cell lines that are tumorigenic following instillation. Intramural injection, on the other hand, is marred by the invasiveness of the procedure and the associated morbidity for the host mouse.
With these shortcomings in mind, we modified previous methods to develop a minimally invasive approach for creating orthotopic bladder cancer xenografts. Using ultrasound guidance we have successfully performed percutaneous inoculation of the bladder cancer cell lines UM-UC1, UM-UC3 and UM-UC13 into 50 athymic nude. We have been able to demonstrate that this approach is time efficient, precise and safe. With this technique, initially a space is created under the bladder mucosa with PBS, and tumor cells are then injected into this space in a second step. Tumor growth is monitored at regular intervals with bioluminescence imaging and ultrasound. The average tumor volumes increased steadily in in all but one of our 50 mice over the study period.
In our institution, this novel approach, which allows bladder cancer xenograft inoculation in a minimally-invasive, rapid and highly precise way, has replaced the traditional model.

The established technique to inoculate primary invasive orthotopic bladder cancer xenografts requires laparotomy and mobilization of the bladder. This procedure inflicts significant morbidity on the mice, is technically challenging and time-consuming. We therefore developed a high-precision, percutaneous approach utilizing ultrasound guidance.

微创建立小鼠原位膀胱移植瘤的

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

在脑肿瘤和关节炎的用荧光SAPC-DOPS纳米囊泡体光学成像

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

Fast and Accurate Exhaled Breath Ammonia Measurement

This exhaled breath ammonia method uses a fast and highly sensitive spectroscopic method known as quartz enhanced photoacoustic spectroscopy (QEPAS) that uses a quantum cascade based laser. The monitor is coupled to a sampler that measures mouth pressure and carbon dioxide. The system is temperature controlled and specifically designed to address the reactivity of this compound. The sampler provides immediate feedback to the subject and the technician on the quality of the breath effort. Together with the quick response time of the monitor, this system is capable of accurately measuring exhaled breath ammonia representative of deep lung systemic levels. 
Because the system is easy to use and produces real time results, it has enabled experiments to identify factors that influence measurements. For example, mouth rinse and oral pH reproducibly and significantly affect results and therefore must be controlled. Temperature and mode of breathing are other examples. As our understanding of these factors evolves, error is reduced, and clinical studies become more meaningful. This system is very reliable and individual measurements are inexpensive. 
The sampler is relatively inexpensive and quite portable, but the monitor is neither. This limits options for some clinical studies and provides rational for future innovations.

Ammonia is an important physiologic metabolite relevant to various disease and wellness states. It is also a difficult molecule to measure in breath, which demands particular precautions be taken to obtain accurate results. Not all factors influencing ammonia are known, but progress can be difficult without accounting for these factors.

快速，准确的呼出气测量氨

This exhaled breath ammonia method uses a fast and highly sensitive spectroscopic method known as quartz enhanced photoacoustic spectroscopy (QEPAS) that ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

从人类肿瘤异种移植的小鼠细胞的消耗显着改善靶细胞下游分析

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...