We would like to thank Rajwant Kaur for technical assistance. We would like to thank Maxwell Tom for assistance with lentivirus preparation. This work was supported by the Reza and Georgianna Khatib Endowed Chair in Skull Base Tumor Surgery at UCSF, and the Michael J. Marchese Professor and Chair at Northwestern University.

下面描述的所有程序已经审查和批准的机构动物使用及护理委员会在西北大学，并已在无菌条件下进行。 1.修改GL261细胞与萤火虫荧光素酶记者表达的注:HIV-1的基于慢病毒载体表达的脾脏病灶形成病毒（SFFV）启动子的控制下，萤火虫荧光素酶（Fluc）。光学报道基因被克隆到载体质粒pHRSIN-CSGW-dlNotI。 <ol><li>在含有95的潮湿气氛维持293-T细胞在补充有10％胎牛血清（FBS）和GL261细胞在补充有10％FBS和1％青霉素 - 链霉素的RPMI培养基中的Dulbecco改进的Eagle培养基（DMEM）中于37℃下％空气和5％二氧化碳（CO 2）。 </li><li>当293-T细胞培养物达到80％汇合在T-75烧瓶中，与pH值一次洗细胞osphate缓冲盐水（PBS）无Ca2 +和Mg2 +，通过保持该烧瓶稍微倾斜孵育细胞用3ml胰蛋白酶（0.05％）的在37℃下5分钟，磨碎用5毫升吸管和收集所有的细胞从烧瓶底部。传输3毫升胰蛋白酶细胞悬浮液入15ml锥形管中，并加入7 ml的肉RPMI培养基（共10毫升细胞悬浮液）的。 </li><li>用5毫升吸管混合细胞悬浮液很好。取100微升从管中的细胞悬浮液和计数用血球细胞的数目。为此，混合100微升的细胞悬浮液用300μl的台盼蓝溶液和插入2微升混合物成血球。计数细胞的数目，而不脱离在显微镜下四个独立的观点蓝染色。平均细胞数目从每个视图，根据该数代表10 4细胞/ ml。 </li><li>同时，离心含有细胞悬浮液30中的管0×g离心7分钟。吸媒体和重悬细胞沉淀用新鲜的RPMI培养基使一个GL261细胞悬浮液以1.0×10 6 / ml的密度。种子2.5×10 6个细胞GL261用2.5ml RPMI培养基中到带有T-175烧瓶中27.5毫升DMEM（1天）。 </li><li> 24小时后，更换用20ml新鲜的DMEM（2天）的介质。 </li><li>以产生慢病毒载体，混合54微升转染试剂用2ml无血清DMEM 5分钟。加入3微克的gag-POL的，3微克水泡性口炎病毒G包膜（VSV-G），以及4.5微克Fluc（PSIN-Fluc）的与转染试剂的培养基，孵育20分钟，在室温。删除整个DNA混合物到293-T的烧瓶（T-175），并在含95％空气和5％CO 2的48小时（2天）的潮湿室中在37℃。 </li><li>加入10ml新鲜的DMEM在24小时转染后（293-T细胞培养基总体积应为大约30毫升）（第3天）。 </li><li>要拆分GL261细胞成24孔板，一次用PBS通过保持烧瓶稍微倾斜洗细胞无Ca2 +和Mg2 +，孵育细胞用5ml胰蛋白酶（0.05％）的在37℃下5分钟，磨碎用5毫升吸管，收集所有的细胞从烧瓶底部，并且计数通过血球细胞如在步骤1.2。同时，离心管中，在300×g离心7分钟。吸媒体和重悬细胞沉淀用新鲜的RPMI培养基使一个GL261细胞悬浮液以2.5×10 4个/ ml的密度。种子2.5×10 4个细胞GL261用1ml RPMI培养基中在24孔板（第3天）。 </li><li>收集30 ml的慢病毒上清液，用10ml移液管从293-T的烧瓶（T-175）在48小时转染后，将上清液转移到50ml锥形管中。抽吸通过在50ml的注射器30 ml的慢病毒上清液，通过0.22μm注射器式滤器，并分装病毒上清进1ml等份（4天）。 </li><li>更换1ml的的R在GL261单元板（24孔）用1ml的慢病毒上清液和PMI介质，在37℃孵育，在湿润室（4天）。 </li><li> 48小时后的慢病毒感染的，用1毫升新鲜的RPMI（6天）更换培养基。 </li><li> 24小时后，重复GL261细胞（7天）的慢病毒感染。 </li><li>后第 2次48小时的慢病毒感染的，用1毫升新鲜的RPMI（9天）更换培养基。 </li><li>持续增长的萤光素酶改性GL261细胞（被称为GL261.luc细胞）和扩大细胞培养到T-75烧瓶中。 </li><li>当GL261.luc细胞培养物达到70％汇合在T-75烧瓶中，通过胰蛋白酶消化收获细胞并通过血细胞计数如在步骤1.2计数。板GL261.luc细胞到24孔板中于毕业密度（1.0×10 6，5.0×10 5，2.5×10 5，1.0×10 5，5.0×10 4，2.5×10 4个细胞/孔）和在一个加湿器孵育在37℃下田间室含95％空气和5％CO 2的3小时。 </li><li>测量荧光素酶活性相对蜂窝到荧光素酶变 ​​形的U-87 MG（U87.luc），使用生物发光成像（图1）的细胞。 <ol><li>启动图像处理软件（点击桌面上的图标），并初始化成像系统（点击"初始化"按钮）。初始化会自动开始并可能需要几分钟的系统检查和相机冷却至-90℃。 </li><li>添加25微升荧光素（D-萤光素钾盐，150毫克/千克）到每个孔中。 </li><li>孵育细胞荧光素为1分钟，在RT和图像的板为10秒。要设置成像系统，选择"发光"，"10秒"的曝光时间，而"中"分档。将24孔板在成像站上，并点击"获取"按钮，开始图像。 </li><li>成功后获得的图像，你会看到一个图像窗口和工具PA莱特在屏幕上。点击"ROI（感兴趣区域）的工具"，并从该狭缝图标选择6×4。创建6×4的狭缝，以覆盖24孔板的图像上的信号的区域，并点击测量标签（铅笔图标）。观察用ROI的测量结果的表中的一个窗口单元每每平方厘米（光子/秒/ SR / cm 2）的球面度第二光子。 </li><li>使用U87.luc细胞预先用这里用于GL261 变形例11相同的慢病毒改性，作为评估在转导的细胞GL261.luc萤光素酶活性的控制。 </li></ol></li></ol> 2.在体外增殖试验<ol><li>当GL261和GL261.luc细胞培养物达到70％汇合在T-75烧瓶中，通过胰蛋白酶消化收获两种细胞系和计数作为在步骤1.2，和板的细胞在96孔板中于1500个细胞的密度80微升每使用以及多道移液器，以减少时间和移液错误RPMI培养基。硒编每个细胞系成20个井总。为了限制在填充的细胞的孔的蒸发，填充空孔用100μl的新鲜RPMI培养基。 </li><li>评估用比色细胞增殖测定的细胞增殖。在这里，我们使用MTS细胞增殖试验。使用多通道移液管，添加20微升的MTS试剂入96孔板，包含细胞的每个孔中。 </li><li>孵育板在37℃下在含95％空气和5％CO 2的3小时的潮湿室中。用酶标仪测量490nm处的吸光度。这被重复，在天1，2，4，和7，电镀后。为了标准化吸光度值，每一天的值由在第1天（图2）获得的相应读数除以。 </li></ol> 3.肿瘤细胞移植注意:所有高压灭菌设备。通过喷洒消毒剂（2％洗必泰溶液）准备手术区，盖上absorben吨窗帘。为了保持无菌条件下，在手术过程中穿无菌手术手套，并划分手术区到由彩色带的两个领域。区域1标有"清洁"，并包含消毒用品;区域2被标记为"脏"，其中包含用于材料。 <ol><li>前动物手术，制备细胞悬浮液为颅内注射。从70％汇合的T-75烧瓶中通过胰蛋白酶消化并计数通过血球细胞如在步骤1.2，使细胞悬浮液以1.0×10 5个细胞/微升在Hanks&#39;平衡盐溶液的密度不含Ca 2+和收获细胞镁离子（HBSS）。 </li><li>麻醉小鼠腹膜内注射含0.9％盐氯胺酮甲苯噻嗪的100 mg / kg和10mg / kg的200微升混合物。要确认正确的麻醉，捏老鼠的脚趾。如果鼠标完全麻醉下，鼠标不应响应的刺激。 </li><li>辖0.1米通过皮下注射克/公斤丁丙诺啡，以减轻术后疼痛。 </li><li>剃使用推子和清洁用棉尖涂药蘸2％氯己定溶液的动物的头顶部。应用眼膏手术过程中保持足够的润滑。 </li><li>做一个切口矢状约为1厘米长比使用无菌手术刀顶枕骨。为了显现前囟，擦拭头骨表面用棉尖涂药浸泡在3％的过氧化氢溶液。 </li><li>钻颅骨，用无菌25-G针，使一个小孔3毫米至前囟门的右侧，仅落后于冠状缝。 </li><li>以确保颅内注射部位是3毫米从颅骨的深度，切割3毫米断用剪刀20微升枪头的尖端，并插入一个注射器插入枪头。 </li><li>垂直插入注射器之前创建的孔，慢慢注入肿瘤细胞SUS养老金含有3.0×10 5细胞在3微升HBSS，在1分钟内。留在原地针一分钟，然后慢慢拔出针。 </li><li>涂上棉尖涂药蘸3％双氧水和2％洗必泰溶液的头骨。干燥头骨表面与棉尖涂药。切出的无菌骨蜡约3毫米3大小，附加骨蜡上的棉尖涂药的端侧。应用骨蜡以支付与棉尖涂药的孔中。 </li><li>头皮的每一侧拉在一起过使用镊子颅骨，和订书钉到闭合伤口。清洁用棉尖蘸2％洗必泰溶液的伤口。 </li><li>监视所有小鼠手术后，直到他们成为走动，并保留正常活动。通常情况下，恢复时间约为30分钟。不要将小鼠与其他动物的笼子里，直到它们完全从麻醉中恢复。 </li></ol><ol><li>喷雾消毒剂对干净的纸巾大方量（0.05％二甲基氯化铵溶液）。使用消毒液浸泡过的纸巾彻底抹了黑片，鼻锥和分频器的成像室，在这里的动物将与该装置接触内部。彻底擦拭所有的表面用清洁，干燥的纸巾。重复此一次，并等待5分钟。 </li><li>初始化成像站如步骤1.14.1。 </li><li>麻醉小鼠用含有200微升氯胺酮/甲苯​​噻嗪和100μl萤光素（D-萤光素钾盐，150毫克/千克）通过腹膜内注射300微升混合物。 </li><li>等待10分钟，注射后确认，如果老鼠是完全在麻醉状态下按捏尾巴。要设置成像系统，选择"发光"，"自动"的曝光时间，而"中"分档。将小鼠在成像为static并点击"获取"按钮，开始图像。确保注射后的时间是与每个成像会议一致。 </li><li>成功后获得的图像，图像窗口和工具选项板应该会出现。点击"投资回报工具"，并从圆圈图标中选择"自动"。要定义投资回报，包围图像信号的区域，然后取投资回报率的测量单位为光子/秒/ SR /厘米2。 </li><li>就拿老鼠出来的成像室和监测小鼠，直到他们成为走动，并保留正常活动。通常的恢复时间是约15分钟。不要将小鼠与其他动物的笼子里，直到它们完全从麻醉中恢复。 </li><li>监测肿瘤生长通过生物发光成像每周两次直至动物呈现下列症状:重量损失（相对于20％以上的损失在植入前的重量），食欲不振（完整厌食24小时），和缺乏持续purposefu的●响应，以温和的刺激（两掌起床），此时他们应该被实施安乐死。安乐死的动物与这些症状使用CO 2室颈椎脱位。 <ol><li>将小鼠放入安乐死室（不要人满为患室），并流入压缩二氧化碳为5-6分钟。注意，所有的动物都昏迷，呼吸停止后大约5分钟。 </li><li>就拿老鼠出室。确保心脏没有跳动摸着你的拇指和食指之间的胸部和检查，没有眨眼反射。 </li><li>抑制鼠标在上平坦表面的俯卧位和把握颈部的尾部的后面，在颅底用一只手和碱用拇指和另一方面的第一手指。 </li><li>抑制头部，迅速拉尾巴落后。确保头骨是使用你的拇指和食指的第一颈椎分离。 </li></ol></li><li>规范阿利兹发光值的每一天对在生物发光成像的第一天获得如在步骤2.3（图3A）相应的读数。 </li><li>进行统计分析，生成用Kaplan-Meier估算12生存曲线，并比较使用对数秩检验13存活曲线之间的差异。 </li></ol>

<ol>
	<li>Clark, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurosurgical+management+and+prognosis+of+patients+with+glioblastoma+that+progresses+during+bevacizumab+treatment.">Neurosurgical management and prognosis of patients with glioblastoma that progresses during bevacizumab treatment.</a> Neurosurgery. 70 (2), 361-370 (2012).</li><li>Stupp, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiotherapy+plus+concomitant+and+adjuvant+temozolomide+for+glioblastoma.">Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma.</a> N Engl J Med. 352 (10), 987-996 (2005).</li><li>Grossman, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+of+patients+with+newly+diagnosed+glioblastoma+treated+with+radiation+and+temozolomide+in+research+studies+in+the+United+States.">Survival of patients with newly diagnosed glioblastoma treated with radiation and temozolomide in research studies in the United States.</a> Clin Cancer Res. 16 (8), 2443-2449 (2010).</li><li>Sughrue, M. E., 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=Immunological+considerations+of+modern+animal+models+of+malignant+primary+brain+tumors.">Immunological considerations of modern animal models of malignant primary brain tumors.</a> J Transl Med. 7 (84), (2009).</li><li>Ausman, J. I., Shapiro, W. R., Rall, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+chemotherapy+of+experimental+brain+tumors:+development+of+an+experimental+model.">Studies on the chemotherapy of experimental brain tumors: development of an experimental model.</a> Cancer Res. 30 (9), 2394-2400 (1970).</li><li>Zagzag, D., Zhong, H., Scalzitti, J. M., Laughner, E., Simons, J. W., Semenza, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+hypoxia-inducible+factor+1alpha+in+brain+tumors:+association+with+angiogenesis,+invasion,+and+progression.">Expression of hypoxia-inducible factor 1alpha in brain tumors: association with angiogenesis, invasion, and progression.</a> Cancer. 88 (11), 2606-2618 (2000).</li><li>Cha, 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=Dynamic,+contrast-enhanced+perfusion+MRI+in+mouse+gliomas:+correlation+with+histopathology.">Dynamic, contrast-enhanced perfusion MRI in mouse gliomas: correlation with histopathology.</a> Magn Reson Med. 49 (5), 848-855 (2003).</li><li>Ksendzovsky, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+immunosuppressive+mechanisms+in+a+mouse+glioma+model.">Investigation of immunosuppressive mechanisms in a mouse glioma model.</a> J Neurooncol. 93 (1), 107-114 (2009).</li><li>Biollaz, G., 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=Site-specific+anti-tumor+immunity:+differences+in+DC+function,+TGF-beta+production+and+numbers+of+intratumoral+Foxp3++Treg.">Site-specific anti-tumor immunity: differences in DC function, TGF-beta production and numbers of intratumoral Foxp3+ Treg.</a> Eur J Immunol. 39 (5), 1323-1333 (2009).</li><li>El Andaloussi, A., Han, Y., Lesniak, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prolongation+of+survival+following+depletion+of+CD4+++CD25++regulatory+T+cells+in+mice+with+experimental+brain+tumors.">Prolongation of survival following depletion of CD4 + CD25+ regulatory T cells in mice with experimental brain tumors.</a> J Neurosurg. 105 (3), 430-437 (2006).</li><li>Dinca, E. 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=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 (3), 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, 185207 (1972).</li><li>Armitage, P., Berry, G., Matthews, J. N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Statistical Methods in Medical Research. , (2001).</li><li>Newcomb, E. W., 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=The+combination+of+ionizing+radiation+and+peripheral+vaccination+produces+long-term+survival+of+mice+bearing+established+invasive+GL261+gliomas.">The combination of ionizing radiation and peripheral vaccination produces long-term survival of mice bearing established invasive GL261 gliomas.</a> Clin Cancer Res. 12 (15), 4730-4737 (2006).</li><li>Pellegatta, 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=Neurospheres+enriched+in+cancer+stem-like+cells+are+highly+effective+in+eliciting+a+dendritic+cell-mediated+immune+response+against+malignant+gliomas.">Neurospheres enriched in cancer stem-like cells are highly effective in eliciting a dendritic cell-mediated immune response against malignant gliomas.</a> Cancer Res. 66 (21), 10247-10252 (2006).</li><li>Clark, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+luciferase+expression+does+not+alter+immunologic+or+in+vivo+growth+properties+of+GL261+murine+glioma+cells.">Stable luciferase expression does not alter immunologic or in vivo growth properties of GL261 murine glioma cells.</a> J Transl Med. 12, 345-34 (2014).</li><li>Abdelwahab, M. G., Sankar, T., Preul, M. C., Scheck, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracranial+implantation+with+subsequent+3D+in+vivo+bioluminescent+imaging+of+murine+gliomas.">Intracranial implantation with subsequent 3D in vivo bioluminescent imaging of murine gliomas.</a> J Vis Exp. (57), e3403 (2011).</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>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></ol>

There are no conflicts of interest to disclose.

GL261鼠神经胶质瘤细胞，颅内植入同基因免疫C57BL / 6小鼠时，相比于人神经胶质瘤的异种移植动物模型中提供了若干优点。许多异种移植的肿瘤生长为封装病变不准确概括的蔓延性人类疾病。相比之下，GL261肿瘤不仅展现侵入邻近脑，而且新生血管形成，核分裂象，而深刻坏死7。最重要的是研究肿瘤免疫学或免疫治疗策略15，16中，C57BL / 6小鼠保留完整的免疫系统8时。以往的研究表明免疫抑制细胞因子TGF-β的表达健壮和颅内肿瘤含有免疫抑制的T调节性细胞类似于人类成胶质细胞瘤9,10。 此处所描述的简单的技术允许荧光素酶由GL261细胞稳定表达。我们最近报道西米拉尔在体外和 GL261.luc细胞相比GL261细胞体内生长 ，除了类似的肿瘤组织学特征和免疫细胞渗透17。 GL261.luc细胞稳定表达的荧光素酶可催化底物萤光素化学能转化为光子，因此，可检测的光的氧化。萤光素可以安全地施用于动物和十字架腹膜内或静脉内注射后的血脑屏障。在小型研究动物如小鼠，生物发光可以从外部以非侵入性方式11检测 。因此，肿瘤生长可以被串行评估，而无需牺牲动物或昂贵的MRI或CT成像。 在协议中的关键步骤涉及，不仅慢病毒转导，而且还验证萤光素酶的稳定表达的体外开始任何体内实验之前。传导损失可能requi再重复慢病毒感染。抗生素抗性基因插入到表达载体的引入也可用于选择萤光素酶表达。细致的技术是必需的颅内植入可再现地放置细胞的一致数目以精确的解剖位置，以允许不同的治疗组进行比较。目前的研究和萤光素酶表达GL261细胞以前的研究的主要区别是使用徒手技术用于植入的细胞相比，使用立体定位框架18的。之前我们已经证实一致的植入结果与徒手技术19。自由手技术的优点是，以执行不同的处理剂的高通量分析的能力。在我们的手中，我们能在1小时内植入大约60只动物。 掌握该技术后，该技术的未来的应用是无限的。由于GL261 demonstr茨升高有丝分裂和类似人类成胶质细胞瘤肿瘤生长迅速，抗增殖治疗进行评估。同样，抗侵袭和抗血管生成疗法，可以用作GL261肿瘤侵入和血管生成。评估化疗药物针对人体目标的效果时，一定要小心。相比利用表达荧光素酶20胶质瘤异种移植物的以前的研究中，这将被视为我 ​​们的模型的一个限制。另外，肿瘤生长的免疫治疗策略的效果可以研究在GL261异种移植模型。

Malignant glioma is the most common and most lethal human brain tumor. Even when treated with maximal surgical resection, radiotherapy, and chemotherapy, median survival remains 15-20 months at major brain tumor referral centers1, 2, 3. The most aggressive form of malignant glioma, glioblastoma, is characterized by mitotic figures, neovascularization, invasion into adjacent brain, and pseudopalisading necrosis. Orthotopic brain tumor xenografts using human brain tumor cells have served an essential function in neuro-oncology research and facilitated pre-clinical translation for testing of new agents, prior to entry into human clinical trials. Whereas most of human xenograft models recapitulate many features of the human disease, a fundamental limitation with all human-based xenograft model systems is the use of immunodeficient animals4. 
The absence of an intact host response clearly modifies tumor growth both in terms of tumor morphology, and efficiency of engraftment. While certain assumptions are acceptable in the interpretation of results from xenografted models, achieving relevant conclusions in the area of therapeutic assessment is a challenge. Certain fundamental biologic features are likely to be similar in immunodeficient and immunocompetent animals, but others such as tumor associated inflammation, tissue invasion, response to injury are probably very different. In contrast, GL261 is a chemically induced murine glioma cell line that accurately recapitulates glioma when implanted into the brains of immunocompetent syngeneic C57BL/6 mice5. Similar to the human disease, intracranial tumor growth rapidly and reproducibly leads to neurologic symptoms and animal demise6-10. 
Subcutaneous tumor growth can be directly measured. Intracranial tumor growth can only be measured with animal sacrifice or with costly imaging studies. Stable expression of the enzyme luciferase allows non-invasive and cost-effective intracranial bioluminescence imaging11. Bioluminescence of luciferase transfected GL261 cells correlates well with intracranial tumor growth. We have recently directly compared GL261 to luciferase modified GL261 cells and demonstrated no difference in in vitro growth and invasion, immunologic cytokine profile, in vivo survival of intracranial tumor bearing C57BL/6 mice, or immune cell infiltrate. This technique is applicable to glioblastoma preclinical studies which involve non-invasive monitoring of tumor growth.

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			<td height="26" style="height:27px;width:379px;">D-Luciferin, Potassium Salt</td>
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			<td height="26" style="height:27px;width:379px;">Single Ended Round Tip Swab with Wood handle</td>
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			<td height="26" style="height:27px;width:379px;">Hanks&#39; Balanced Salt Solution without Ca2+ and Mg2+ (HBSS)</td>
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</table>

bioluminescence imaging of an immunocompetent animal model for glioblastoma

In contrast to commonly reported human glioma xenograft animal models, GL261 murine glioma xenografts recapitulate nearly all relevant clinical and histopathologic features of the human disease. When GL261 cells are implanted intracranially in syngeneic C57BL/6 mice, the model has the added advantage of maintaining an intact immune microenvironment. Stable expression of luciferase in GL261 cells allows non-invasive cost effective bioluminescence monitoring of intracranial tumor growth. We have recently demonstrated that luciferase expression in GL261 cells does not affect the tumor growth properties, tumor cell immunomodulatory cytokine expression, infiltration of immune cells into the tumor, or overall survival of animals bearing the intracranial tumor. Therefore, it appears that the GL261 luciferase glioma model can be useful in the study of novel chemotherapeutic and immunotherapeutic modalities. Here we report the technique for generating stable luciferase expression in GL261 cells and how to study the in vitro and in vivo growth of the tumor cells by bioluminescence imaging.

GL261细胞用如步骤如上所述1.在体外生物发光成像演示类似于阳性对照U87.luc人恶性胶质瘤细胞系的水平健壮萤光素酶表达（图1）含有萤光素酶的慢病毒。正如预期的那样，未感染GL261细胞表现出没有背景荧光素酶表达。这一步很简单，但关键的执行之前，用被感染的细胞系，以确认稳定的荧光素酶表达进一步的研究。 颅内植入后荧光素酶的表达。 颅内植入前，我们展示了GL261.luc 体外生长速度没有差异相比，GL261 细胞 （图2）。颅内生长和存活分析，将细胞注射到BR对于生物发光成像使用协议步骤4 C57BL / 6小鼠的荷瘤小鼠的肿瘤GL261.luc AINS按照协议步骤3.肿瘤生长串联分析并证实检测的肿瘤生长（图3A）。荷GL261.luc肿瘤快速和持续成为奄奄一息，被实施安乐死。重要的是，在GL261.luc和GL261荷瘤动物（图3B）之间的总生存没有差别。 体内生物发光成像，因此可以用于监测响应于实验胶质母细胞瘤的治疗方法。快速可靠的肿瘤生长和短总生存所用的时间相对短的量，得到大量的数据。 <img alt="图1" src="/files/ftp_upload/53287/53287fig1.jpg" /> 在GL261.luc细胞图1萤光素酶活性。U87.luc，GL261.luc和GL261（阴性对照）的细胞铺板在密度1.0×10 6，5.0×10 5，2.5×10 5，1.0×10 5，5.0×10 4，2.5×10 4个细胞/孔的由左到右。发光（光子/秒/ SR /厘米2）由管腔成像站测得。 <a href="https://www.jove.com/files/ftp_upload/53287/53287fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53287/53287fig2.jpg" /> 图2.萤光素酶表达不影响GL261细胞增殖。GL261.luc细胞不引起 在增殖差就证明了MTS细胞增殖测定。该图显示增加倍数，相对于1天，如通过比较平均吸光度值来确定（平均值±SEM）在thespecified时间点到在第1天的平均值（比较非配对t检验值每个细胞系之间:P = 0.7796）14。误差棒代表平均值从每天四份样品（平均值±SEM）。 <a href="https://www.jove.com/files/ftp_upload/53287/53287fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53287/53287fig3.jpg" /> 图3.萤光素酶表达剂量不会对体内 肿瘤生长和动物存活的 影响。 （一）生物发光监测表明颅内GL261.luc瘤C57BL / 6小鼠进行性生长。（B）Kaplan-Meier生存分析表明总体生存与任何GL261（实线）或GL261.luc细胞植入颅内的小鼠没有区别（虚线）。请点击此处查看该图的放大版本。

Watch this Scientific Journal Video about Bioluminescence Imaging of an Immunocompetent Animal Model for Glioblastoma at JoVE.com

Bioluminescence Imaging of an Immunocompetent Animal Model for Glioblastoma

GL261 glioma cells provide a useful immunocompetent animal model of glioblastoma. The goals of this protocol are to demonstrate proper techniques for monitoring intracranial tumor growth using in vivo bioluminescence imaging, and to verify the utility of luciferase-modified GL261 cells for studying tumor immunology and immunotherapeutic approaches for treating glioblastoma.

免疫活性动物模型胶质母细胞瘤的生物发光成像

In contrast to commonly reported human glioma xenograft animal models, GL261 murine glioma xenografts recapitulate nearly all relevant clinical and ...

bioluminescence-imaging-an-immunocompetent-animal-model-for

Research

JoVE Journal

Medicine

14.7K 次观看.  University of California San Francisco. GL261 glioma cells provide a useful immunocompetent animal model of glioblastoma. The goals of this protocol are to demonstrate proper techniques for monitoring intracranial tumor growth using in vivo bioluminescence imaging, and to verify the utility of luciferase-modified GL261 cells for studying tumor immunology and immunotherapeutic approaches for treating glioblastoma.

视频: Bioluminescence Imaging of an Immunocompetent Animal Model for Glioblastoma

Monitoring Tumor Metastases and Osteolytic Lesions with Bioluminescence and Micro CT Imaging

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence imaging using IVIS Spectrum was employed to monitor the distribution and development of the tumor cells following the delivery procedure including DLIT reconstruction to measure the tumor signal and its location.
Development of metastatic lesions to the bone tissues triggers osteolytic activity and lesions to tibia and femur were evaluated longitudinally using micro CT. Imaging was performed using a Quantum FX micro CT system with fast imaging and low X-ray dose. The low radiation dose allows multiple imaging sessions to be performed with a cumulative X-ray dosage far below LD50. A mouse imaging shuttle device was used to sequentially image the mice with both IVIS Spectrum and Quantum FX achieving accurate animal positioning in both the bioluminescence and CT images. The optical and CT data sets were co-registered in 3-dimentions using the Living Image 4.1 software. This multi-mode approach allows close monitoring of tumor growth and development simultaneously with osteolytic activity.

An experimental mouse model of bone metastasis was established following intracardiac delivery of luciferase expressing mammary tumor cells. Tumor development and resulted osteolytic lesion were monitored longitudinally with bioluminescence and micro CT imaging.

生物发光和微型CT成像监测肿瘤转移和溶骨性病变

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence ...

科研

医学

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

在小鼠模型体内生物发光成像乳腺癌脑转移的肿瘤乏氧动力学

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

在动物模型揭示神经，血液，创伤后应激障碍和行为相关因素的生物标志物

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Use of Animal Model of Sepsis to Evaluate Novel Herbal Therapies

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several techniques, including infusion of exogenous bacterial toxin (endotoxemia) or bacteria (bacteremia), as well as surgical perforation of the cecum by cecal ligation and puncture (CLP)1-3. CLP allows bacteria spillage and fecal contamination of the peritoneal cavity, mimicking the human clinical disease of perforated appendicitis or diverticulitis. The severity of sepsis, as reflected by the eventual mortality rates, can be controlled surgically by varying the size of the needle used for cecal puncture2. In animals, CLP induces similar, biphasic hemodynamic cardiovascular, metabolic, and immunological responses as observed during the clinical course of human sepsis3. Thus, the CLP model is considered as one of the most clinically relevant models for experimental sepsis1-3.
Various animal models have been used to elucidate the intricate mechanisms underlying the pathogenesis of experimental sepsis. The lethal consequence of sepsis is attributable partly to an excessive accumulation of early cytokines (such as TNF, IL-1 and IFN-&gamma;)4-6 and late proinflammatory mediators (e.g., HMGB1)7. Compared with early proinflammatory cytokines, late-acting mediators have a wider therapeutic window for clinical applications. For instance, delayed administration of HMGB1-neutralizing antibodies beginning 24 hours after CLP, still rescued mice from lethality8,9, establishing HMGB1 as a late mediator of lethal sepsis. The discovery of HMGB1 as a late-acting mediator has initiated a new field of investigation for the development of sepsis therapies using Traditional Chinese Herbal Medicine. In this paper, we describe a procedure of CLP-induced sepsis, and its usage in screening herbal medicine for HMGB1-targeting therapies.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection, and can be simulated by a surgical technique termed cecal ligation and puncture (CLP). Here we describe a method to use CLP-induced animal model to screen medicinal herbs for therapeutic agents.

脓毒症动物模型，用于评估新型草药疗法

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several ...

An Orthotopic Glioblastoma Mouse Model Maintaining Brain Parenchymal Physical Constraints and Suitable for Intravital Two-photon Microscopy

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology rely on the injection of a suspension of glioma cells into the brain parenchyma following incision of the dura-mater. Whereas the cells have to be injected superficially to be accessible to intravital two-photon microscopy, superficial injections fail to recapitulate the physiopathological conditions. Indeed, escaping through the injection tract most tumor cells reach the extra-dural space where they expand abnormally fast in absence of mechanical constraints from the parenchyma.
Our improvements consist not only in focally implanting a glioma spheroid rather than injecting a suspension of glioma cells in the superficial layers of the cerebral cortex but also in clogging the injection site by a cross-linked dextran gel hemi-bead that is glued to the surrounding parenchyma and sealed to dura-mater with cyanoacrylate. Altogether these measures enforce the physiological expansion and infiltration of the tumor cells inside the brain parenchyma. Craniotomy was finally closed with a glass window cemented to the skull to allow chronic imaging over weeks in absence of scar tissue development.
Taking advantage of fluorescent transgenic animals grafted with fluorescent tumor cells we have shown that the dynamics of interactions occurring between glioma cells, neurons (e.g. Thy1-CFP mice) and vasculature (highlighted by an intravenous injection of a fluorescent dye) can be visualized by intravital two-photon microscopy during the progression of the disease.
The possibility to image a tumor at microscopic resolution in a minimally compromised cerebral environment represents an improvement of current GBM animal models which should benefit the field of neuro-oncology and drug testing.

We have established a cortical orthotopic glioblastoma model in mice for intravital two-photon microscopy that recapitulates the biophysical constraints normally at play during the growth of the tumor. A chronic glass window replacing the skull above the tumor enables the follow-up of the tumor progression over time by two-photon microscopy.

原位胶质母细胞瘤小鼠模型的维护脑实质的物理约束，并适用于活体双光子显微镜

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology ...

An Isolated Working Heart System for Large Animal Models

Since its introduction in the late 19th century, the Langendorff isolated heart perfusion apparatus, and the subsequent development of the working heart model, have been invaluable tools for studying cardiovascular function and disease1-15. Although the Langendorff heart preparation can be used for any mammalian heart, most studies involving this apparatus use small animal models (e.g., mouse, rat, and rabbit) due to the increased complexity of systems for larger mammals1,3,11. One major difficulty is ensuring a constant coronary perfusion pressure over a range of different heart sizes &#8211; a key component of any experiment utilizing this device1,11. By replacing the classic hydrostatic afterload column with a centrifugal pump, the Langendorff working heart apparatus described below allows for easy adjustment and tight regulation of perfusion pressures, meaning the same set-up can be used for various species or heart sizes. Furthermore, this configuration can also seamlessly switch between constant pressure or constant flow during reperfusion, depending on the user&#8217;s preferences. The open nature of this setup, despite making temperature regulation more difficult than other designs, allows for easy collection of effluent and ventricular pressure-volume data.

Most studies involving the Langendorff apparatus use small animal models due to the increased complexity of systems for larger mammals. We describe a Langendorff system for large animal models that allows for use across a range of species, including humans, and relatively easy data acquisition.

一个孤立的工作心脏系统的大型动物模型

Since its introduction in the late 19th century, the Langendorff isolated heart perfusion apparatus, and the subsequent development of the working heart ...

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans biofilms have been studied predominantly in vitro but there is a crucial need for better understanding of this dynamic process under in vivo conditions. We developed an in vivo subcutaneous rat model to study C. albicans biofilm formation. In our model, multiple (up to 9) Candida-infected devices are implanted to the back part of the animal. This gives us a major advantage over the central venous catheter model system as it allows us to study several independent biofilms in one animal. Recently, we adapted this model to study C. albicans biofilm development in BALB/c mice. In this model, mature C. albicans biofilms develop within 48 hr and demonstrate the typical three-dimensional biofilm architecture. The quantification of fungal biofilm is traditionally analyzed post mortem and requires host sacrifice. Because this requires the use of many animals to perform kinetic studies, we applied non-invasive bioluminescence imaging (BLI) to longitudinally follow up in vivo mature C. albicans biofilms developing in our subcutaneous model. C. albicans cells were engineered to express the Gaussia princeps luciferase gene (gLuc) attached to the cell wall. The bioluminescence signal is produced by the luciferase that converts the added substrate coelenterazine into light that can be measured. The BLI signal resembled cell counts obtained from explanted catheters. Non-invasive imaging for quantifying in vivo biofilm formation provides immediate applications for the screening and validation of antifungal drugs under in vivo conditions, as well as for studies based on host-pathogen interactions, hereby contributing to a better understanding of the pathogenesis of catheter-associated infections.

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

We present an experimental procedure of Candida albicans biofilm development in a mouse subcutaneous model. Fungal biofilms were quantified by determining the number of colony forming units and by a non-invasive bioluminescence imaging, where the amount of light that is produced corresponds with the number of viable cells.

白色念珠菌生物膜发展在医学上，国外相关机构在小鼠皮下型号其次是生物发光成像

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans ...

A Chronic Cardiac Ischemia Model in Swine Using an Ameroid Constrictor

Cardiovascular disease remains the number one cause of mortality in the United States. There are numerous approaches to treating these diseases, but regardless of the approach, an in vivo model is needed to test each treatment. The pig is one of the most used large animal models for cardiovascular disease. Its heart is very similar in anatomy and function to that of a human. The ameroid placement technique creates an ischemic area of the heart, which has many useful applications in studying myocardial infarction. This model has been used for surgical research, pharmaceutical studies, imaging techniques, and cell therapies.
There are several ways of inducing an ischemic area in the heart. Each has its advantages and disadvantages, but the placement of an ameroid constrictor remains the most widely used technique. The main advantages to using the ameroid are its prevalence in existing research, its availability in various sizes to accommodate the anatomy and size of the vessel to be constricted, the surgery is a relatively simple procedure, and the post-operative monitoring is minimal, since there are no external devices to maintain. This paper provides a detailed overview of the proper technique for the placement of the ameroid constrictor.

The purpose of this protocol is to demonstrate the placement of a delayed constricting device (an ameroid constrictor) around a coronary artery in a swine model. This device creates an ischemic area of the heart that is useful for studying new diagnostic imaging techniques and new methods of treatment.

应用 Ameroid 的猪慢性心肌缺血模型

Cardiovascular disease remains the number one cause of mortality in the United States. There are numerous approaches to treating these diseases, but ...

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

There is a significant shortage of liver allografts available for transplantation, and in response the donor criteria have been expanded. As a result, normothermic ex vivo liver perfusion (NEVLP) has been introduced as a method to evaluate and modify organ function. NEVLP has many advantages in comparison to hypothermic and subnormothermic perfusion including reduced preservation injury, restoration of normal organ function under physiologic conditions, assessment of organ performance, and as a platform for organ repair, remodeling, and modification. Both murine and porcine NEVLP models have been described. We demonstrate a rat model of NEVLP and use this model to show one of its important applications &#8212; the use of a therapeutic molecule added to liver perfusate. Catalase is an endogenous reactive oxygen species (ROS) scavenger and has been demonstrated to decrease ischemia-reperfusion in the eye, brain, and lung. Pegylation has been shown to target catalase to the endothelium. Here, we added pegylated-catalase (PEG-CAT) to the base perfusate and demonstrated its ability to mitigate liver preservation injury. An advantage of our rodent NEVLP model is that it is inexpensive in comparison to larger animal models. A limitation of this study is that it does not currently include post-perfusion liver transplantation. Therefore, prediction of the function of the organ post-transplantation cannot be made with certainty. However, the rat liver transplant model is well established and certainly could be used in conjunction with this model. In conclusion, we have demonstrated an inexpensive, simple, easily replicable NEVLP model using rats. Applications of this model can include testing novel perfusates and perfusate additives, testing software designed for organ evaluation, and experiments designed to repair organs.

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

There is a significant liver donor shortage, and criteria for liver donors have been expanded. Normothermic ex vivo liver perfusion (NEVLP) has been developed to evaluate and modify organ function. This study demonstrates a rat model of NEVLP and tests the ability of pegylated-catalase, to mitigate liver preservation injury.

体外常温肝灌注的小动物模型

There is a significant shortage of liver allografts available for transplantation, and in response the donor criteria have been expanded. As a result, ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...