作者想感谢的援助，在发展中国家这种模式Verlene亨利和Lindsay霍尔姆斯。这项工作是部分资金由詹姆斯S麦克唐纳基金会（＃220020173）。

1。细胞系<ol><li>在大型组织，辅以5％胎牛血清（FBS），DMEM - F12文化烧瓶的U87MG胶质瘤细胞培养。 </li><li>细胞是由磷酸用温水冲洗烧瓶两次收获缓冲液（PBS）和孵化，在37 ° C时5分钟10毫升的PBS，含0.25％胰蛋白酶和0.05％EDTA。一旦细胞被取消，他们是文化传媒和离心（4分钟）300 X克含10毫升到50毫升的管。 </li><li>洗净后，细胞悬浮在浓度为10 × 10 6 /毫升培养基，允许50,000个细胞/ 5μL接种。 </li><li>细胞被保存在冰直到颅内注射。 </li></ol> 2。丝杠颅内抽薹 此过程可进行数天前的细胞注射。这里描述的所有程序已经进行了严格的无菌条件下。 <ol><li>编号小鼠（BALB / C NU / NU女性; 5-6周;约18克）是用于识别目的，称重并与腹腔注射氯胺酮（100毫克/公斤）和甲苯噻嗪（5毫克的混合物（IP）的anesthetised /公斤）。 </li><li>碘溶液擦拭皮肤，沿中线和前双耳线右侧小切口（2-3毫米）。这暴露颅骨冠状面和矢状面的缝合。前囟门是定位在这两个缝线交界处。 </li><li>丝杠的切入点，然后标记在一个点上侧2.5毫米和1毫米前的前囟。这点是位于正上方的尾状核1。 </li><li> 1 × 1毫米的深孔钻举行麻花钻硬脑膜通过头骨无菌手。 </li><li>成孔，用螺丝刀将延长到硬脑膜的头骨表面以下 1.6毫米的消毒丝杠然后狂奔，直到冲洗的头骨（图1A ）。 </li><li>无菌钢丝或螺丝假人，然后放置到丝杠的中心孔关闭开幕（图1B）。 </li><li> Vetbond组织粘合剂（N -氰基丙烯酸正丁酯）和小鼠封闭伤口reversine腹腔注射液（小动物）（0.1毫升/公斤）和卡洛芬镇痛（5 mg/kg/100μL）。小鼠变暖垫（36℃），这可能需要长达20分钟，然后让其恢复。老鼠经常在此期间恢复时间的监测和观察，直至他们完全清醒。 </li></ol> 3。颅内细胞植入<ol><li>无菌铐汉密尔顿注射器准备用一个小塑料环，只允许2毫米的针头，针头，延长丝杠插座（图1B）以下。最后的接种点，因此颅骨表面下3.5毫米。 </li><li>丝杠手术后四天，老鼠再次麻醉上述（2.1）和小切口是在丝杠删除的钢丝。 </li><li> 5μL以及采取预防措施，不能创造或制定任何气泡混合细胞充满铐汉密尔顿注射器。 </li><li>固定灌注泵注射器和针头插入到丝杠（图1C）。细胞，然后注入每小时30μL的速度。 </li><li>自动化设备（图1D）允许我们在一个恒定的流量的时候，注入10只动物。细胞也可手动注射，所使用的手铐汉密尔顿注射器或注射器可以由200μL枪头已3毫米修剪，使针尖延长2毫米，超出丝杠铐。注入，然后必须在一个恒定的稳健的步伐。 </li><li>当输液完成后，小心取下注射器和钢丝更换成丝杠。封闭伤口粘和小鼠前（2.7）恢复的药物，并允许恢复变暖垫。 </li></ol> 4。治疗的挑战<ol><li> 4天的U87MG细胞接种后，小鼠称重，随机分为对照组和治疗组。 </li><li>对照组给予PBS（100μL）腹腔（IP）的注射，而治疗组给予AMG102（100微克，在100μL的PBS）的IP注射。 </li><li>重复注射每隔一天为14天，共6注射（图2）。 </li><li>最后的注射后，老鼠每天监测和权衡每隔一天。 </li><li>当小鼠开始显示显著的神经功能障碍（平衡紊乱，瘫痪），脱水，或超过10％的重量损失或垂死的，他们被人道人道毁灭。这些人性化的终点天，然后记录Kaplan - Meier生存曲线。 </li></ol> 5。治疗效率的评价</p&gt; <ol><li> Kaplan - Meier生存曲线生成使用GraphPad棱镜4.03为Windows; GraphPad软件，圣地亚哥美国加利福尼亚州， <a href="http://www.graphpad.com">www.graphpad.com</a> 3。死亡或euthanizations标记为终点事件，得分为1。在实验结束后还活着的动物仍然被作为非活动记录，得分为0。 </li></ol> 6。代表性的成果: 控制动物开始后显示的初始接种细胞，神经系统紊乱和减肥23天的迹象。这是在35天的AMG102组显着延迟。事实上，35天，77％（N = 7 / 9），对照组已安乐死。 Kaplan - Meier生存曲线清楚地表明AMG102治疗（图3）的存活率显着增加。 70天，对照组小鼠的88％已被实施安乐死（N = 8 / 9）相比，37％（N = 3 / 8）AMG102治疗组。大脑的组织学分析证实，在对照组的全部9个动物开发的肿瘤相比，只有8 AMG102治疗的小鼠（图4）3。 <img alt="图1" src="/files/ftp_upload/3157/3157fig1.jpg" /> 图1:图像（a）显示在上方位于尾状核的区域定位颅内丝杠。铐汉密尔顿注射器（与细胞预装），然后放置内丝杠2毫米的针头，使超出指导和尾状核细胞内注入3.5毫米。注射器后取出，取而代之的是一个钢丝（二）。自动输液器（C）可以注入10只动物在同一时间。这里展示的是五（四）同时注射。 <img alt="图2" src="/files/ftp_upload/3157/3157fig2.jpg" /> 图2:原理的研究协议。 0天细胞注射治疗第4天开始。对照组动物注射PBS（100μL）的IP，而治疗组接受注射的AMG102（100μg/100μL）IP。这些注射，每隔一天为10天。一共有6次注射。动物被监测的70天的神经系统和物理扰动的迹象。 <img alt="图3" src="/files/ftp_upload/3157/3157fig3.jpg" /> 图3:Kaplan - Meier生存曲线比较控制和治疗的小鼠AMG102。 AMG102（P = 0.005），治疗后获得显著的生存。 <img alt="图4" src="/files/ftp_upload/3157/3157fig4.jpg" /> 图4:显微冠状脑肿瘤的发展，在控制动物（一）在AMG102治疗的动物和无脑肿瘤的发展（二）部分展示。比例尺为1毫米。

<ol>
	<li>Lal, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+implantable+guide-screw+system+for+brain+tumor+studies+in+small+animals.">An implantable guide-screw system for brain tumor studies in small animals.</a> J Neurosurg. 92, 326-333 (2000).</li><li>Jun, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMG+102,+a+fully+human+anti-hepatocyte+growth+factor/scatter+factor+neutralizing+antibody,+enhances+the+efficacy+of+temozolomide+or+docetaxel+in+U-87+MG+cells+and+xenografts.">AMG 102, a fully human anti-hepatocyte growth factor/scatter factor neutralizing antibody, enhances the efficacy of temozolomide or docetaxel in U-87 MG cells and xenografts.</a> Clin Cancer Res. 13, 6735-6742 (2007).</li><li>Pillay, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+plasticity+of+oncogene+addiction:+implications+for+targeted+therapies+directed+to+receptor+tyrosine+kinases.">The plasticity of oncogene addiction: implications for targeted therapies directed to receptor tyrosine kinases.</a> Neoplasia. 11, 448-458 (2009).</li><li>Buchanan, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiosensitization+of+glioma+cells+by+modulation+of+Met+signaling+with+the+hepatocyte+growth+factor+neutralizing+antibody.+AMG102.">Radiosensitization of glioma cells by modulation of Met signaling with the hepatocyte growth factor neutralizing antibody. AMG102.</a> J Cell Mol Med. 15, 1999-2006 (2011).</li><li>Wen, P. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+phase+II+study+evaluating+the+efficacy+and+safety+of+AMG+102+(rilotumumab)+in+patients+with+recurrent+glioblastoma.">A phase II study evaluating the efficacy and safety of AMG 102 (rilotumumab) in patients with recurrent glioblastoma.</a> Neuro Oncol. 13, 437-446 (2011).</li><li>Caretti, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+of+Tumor+Growth+and+Post-Irradiation+Recurrence+in+a+Diffuse+Intrinsic+Pontine+Glioma+Mouse+Model.">Monitoring of Tumor Growth and Post-Irradiation Recurrence in a Diffuse Intrinsic Pontine Glioma Mouse Model.</a> Brain Pathol. 4, 441-451 (2011).</li><li>Jallo, G. I., Volkov, A., Wong, C., Carson, B. S., Penno, 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=A+novel+brainstem+tumor+model:+functional+and+histopathological+characterization.">A novel brainstem tumor model: functional and histopathological characterization.</a> Childs Nerv. Syst. 22, 1519-1525 (2006).</li><li>Johns, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+efficacy+of+epidermal+growth+factor+receptor-specific+antibodies+against+gliomaxenografts+is+influenced+by+receptor+levels,+activation+status,+and+heterodimerization.">The efficacy of epidermal growth factor receptor-specific antibodies against gliomaxenografts is influenced by receptor levels, activation status, and heterodimerization.</a> Clin Cancer Res. 13, 1911-1925 (2007).</li><li>Scott, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+phase+I+clinical+trial+with+monoclonal+antibody+ch806+targeting+transitional+state+and+mutant+epidermal+growth+factor+receptors.">A phase I clinical trial with monoclonal antibody ch806 targeting transitional state and mutant epidermal growth factor receptors.</a> Proc. Natl. Acad. Sci. U. S. A. 104, 4071-4076 (2007).</li><li>Niyazi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+options+for+recurrent+malignant+glioma.">Therapeutic options for recurrent malignant glioma.</a> Radiother. Oncol. 98, 1-14 (2011).</li></ol>

这项工作是支持由安进公司从一个研究资助

这里介绍的方法，使颅内丝杠快速，重现性颅内移植瘤的建立和一个受过训练的动物技术人员手中，这个过程是很容易没有立体定向设备的需要的情况下进行。我们的方法结合使用一个丝杠，准确地找到最适合在体内肿瘤的发展的大脑区域，并自动输液泵，允许多达10只动物同时接种一次。丝杠隆胸过程只需不到5分钟执行。细胞在30μL/小时的自动输液需要5μL体积仅有10分钟。这将确保以恒定速率准确的剂量和减少额外的颅区，由于颅内压增高的细胞被驱逐的风险。 10μL卷，我们也执行此过程，并没有观察到任何额外的颅内肿瘤的生长，由于细胞回流。至于自动输液泵还允许几种动物同时接种，我们可以注入高达每小时60动物相比，一个立体的过程，是有限的，以每小时约15％的动物。 在此过程中最关键的一步是在颅骨的指导螺丝的位置，作为它的位置决定了肿瘤体积的最终位置。如果丝杠过中央，细胞会被注入到地区之间的两个大脑半球，而如果引导有条件向前，细胞将不会被注入到大脑的。因此，必须在尽可能准确定位指南2.5毫米的横向和1毫米，前囟 1 。在这项研究中，我们使用了钢丝杠，虽然塑料导向螺钉也可用于这些研究需要脑成像如MRI或PET 6 。即使自动输液泵的成本对某些群体望而却步，细胞仍然可以被注入速度比手动更立体定向的方法。总体而言，这种快速，准确，重现性的方法可用于研究脑胶质瘤，髓母细胞瘤和脑干肿瘤 7 。我们两国之间由于意外麻醉反应（&lt;1％）死亡的实验室，超过500只已执行此过程。小鼠，狂奔与丝杠，并随后注入与细胞没有死亡或遭受任何神经系统并发症，作为一个过程的结果。动物存活过去的100天，无任何并发症。虽然我们倾向于使用像头发的SCID一个无毛小鼠，免疫能力受损的老鼠会一样，适当提供头骨头发前去除手术，以保持工地清洁，防止头发干扰丝杠。 有几个为脑胶质瘤8-10的治疗目前正在制订的新疗法。最近，我们发现，皮下的U87MG胶质瘤异种移植可以治疗AMG102 3的抑制作用;向肝细胞生长因子，目前正在进行5胶质瘤临床评价一个人源化抗体。我们所采用的方法在这里描述的，建立在裸小鼠的大脑颅内的U87MG肿瘤。我们的研究清楚地表明该方法的实用工具和演示，AMG102显著抑制原位的U87MG移植瘤的生长。我们将继续使用这个模型来确定与AMG102可使用其他疗法相结合，以获得更有效地抑制肿瘤生长。

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td> DMEM/F12 </td><td> Invitrogen公司</td><td> 12634010 </td><td></td></tr><tr><td> FCS的</td><td> Bovogen </td><td> SFB的</td><td></td></tr><tr><td>胰蛋白酶</td><td> Invitrogen公司</td><td> 15050065 </td><td></td></tr><tr><td> EDTA的</td><td>西格玛</td><td> E6758 </td><td></td></tr><tr><td> BALB / C NU / nu小鼠</td><td> ARC珀斯</td><td></td><td></td></tr><tr><td>螺丝座</td><td>塑料之一</td><td> SD - 1 </td><td></td></tr><tr><td>钻架</td><td>塑料之一</td><td> DH - 1 </td><td></td></tr><tr><td>钻头</td><td>塑料之一</td><td> D＃60 </td><td></td></tr><tr><td>螺杆指南（金属） </td><td>塑料之一</td><td> C212SG </td><td></td></tr><tr><td>螺杆假人（针式） </td><td>塑料之一</td><td> C212SD </td><td></td></tr><tr><td>汉密尔顿注射器（10μL26克凝成针） </td><td>宏力科探索科学</td><td> 80075 </td><td></td></tr><tr><td> PHD 22/2000输液泵</td><td>哈佛仪器</td><td> 70-2003 </td><td>可选</td></tr><tr><td> Vetbond </td><td> 3M </td><td> 1469SB </td><td></td></tr><tr><td>热垫</td><td>哈佛仪器</td><td> 340390 </td><td></td></tr></tbody></table>

a simple guide screw method for intracranial xenograft studies in mice

人类肿瘤细胞移植到免疫抑制小鼠的大脑的脑肿瘤，包括胶质母细胞瘤（神经胶质瘤）和髓母细胞瘤的研究建立的方法。广泛应用于立体定向的方法只允许注射一次一个单一的动物，是劳动密集型的，需要高度专业化的设备。丝杠的方法，最初由拉尔等人，1开发的开发，以消除繁琐的立体程序。我们现在介绍一个修改后的丝杠的方法，是迅速的和极其安全;这两者都是重要的伦理方面的考虑。值得注意的是，现在我们的程序采用输液泵，允许多达10个同时与肿瘤细胞注入动物。 为了演示此过程中的效用，我们确立了颅内移植瘤小鼠，然后用AMG102治疗人类的U87MG胶质瘤细胞向肝细胞生长因子/分散因子，目前正在进行临床评价2-5的完全人类抗体。全身注射的AMG102显著延长所有颅内U87MG移植瘤小鼠的生存，并导致一个完整的治疗方法。 这项研究表明，丝杠方法是一种廉价的，高度重现建立颅内移植瘤的方法。此外，它提供了一个用于验证新的治疗策略，治疗脑癌的有关的生理模型。

Watch this Scientific Journal Video about A Simple Guide Screw Method for Intracranial Xenograft Studies in Mice at JoVE.com

A Simple Guide Screw Method for Intracranial Xenograft Studies in Mice

为了评估新的治疗脑胶质瘤的治疗范式，生理相关的模型是必不可少的。颅内移植瘤模型的建立，是比立体定向的方法更快速，更安全，我们利用一个植入式丝杠程序。

一个简单的指南螺杆颅内移植瘤小鼠的研究方法

The grafting of human tumor cells into the brain of immunosuppressed mice is an established method for the study of brain cancers including glioblastoma ...

a-simple-guide-screw-method-for-intracranial-xenograft-studies-in-mice

Research

JoVE Journal

Medicine

23.0K 次观看.  Monash Institute of Medical Research. 为了评估新的治疗脑胶质瘤的治疗范式，生理相关的模型是必不可少的。颅内移植瘤模型的建立，是比立体定向的方法更快速，更安全，我们利用一个植入式丝杠程序。

视频: A Simple Guide Screw Method for Intracranial Xenograft Studies in Mice

Guide Wire Assisted Catheterization and Colored Dye Injection for Vascular Mapping of Monochorionic Twin Placentas

Monochorionic (MC) twin pregnancies are associated with significantly higher morbidity and mortality rates than dichorionic twins. Approximately 50% of MC twin pregnancies develop complications arising from the shared placenta and associated vascular connections1. Severe twin-to-twin syndrome (TTTS) is reported to account for approximately 20% of these complications2,3. Inter-twin vascular connections occur in almost all MC placentas and are related to the prognosis and outcome of these high-risk twin pregnancies. The number, size and type of connections have been implicated in the development of TTTS and other MC twin conditions. Three types of inter-twin vascular connections occur: 1) artery to vein connections (AVs) in which a branch artery carrying deoxygenated blood from one twin courses along the fetal surface of the placenta and dives into a placental cotyledon. Blood flows via a deep intraparenchymal capillary network into a draining vein that emerges at the fetal surface of the placenta and brings oxygenated blood toward the other twin. There is unidirectional flow from the twin supplying the afferent artery toward the twin receiving the efferent vein; 2) artery to artery connections (AAs) in which a branch artery from each twin meets directly on the superficial placental surface resulting in a vessel with pulsatile bidirectional flow, and 3) vein to vein connections (VVs) in which a branch vein from each twin meets directly on the superficial placental surface allowing low pressure bidirectional flow. In utero obstetric sonography with targeted Doppler interrogation has been used to identify the presence of AV and AA connections4. Prenatally detected AAs that have been confirmed by postnatal placental injection studies have been shown to be associated with an improved prognosis for both twins5. Furthermore, fetoscopic laser ablation of inter-twin vascular connections on the fetal surface of the shared placenta is now the preferred treatment for early, severe TTTS. 
Postnatal placental injection studies provide a valuable method to confirm the accuracy of prenatal Doppler ultrasound findings and the efficacy of fetal laser therapy6. Using colored dyes separately hand-injected into the arterial and venous circulations of each twin, the technique highlights and delineates AVs, AAs, and VVs. This definitive demonstration of MC placental vascular anatomy may then be correlated with Doppler ultrasound findings and neonatal outcome to enhance our understanding of the pathophysiology of MC twinning and its sequelae. Here we demonstrate our placental injection technique.

Vascular mapping of monochorionic (MC) twin placentas after birth provides a means for detailed demonstration of vascular connections between the twins&#x2019; circulations. 
Imbalance of these connections is thought to play a pivotal role in the development of complications of MC twinning including twin-to-twin transfusion syndrome.

导丝辅助导管和彩色染料注射的双人单绒毛膜胎盘血管映射

Monochorionic (MC) twin pregnancies are associated with significantly higher morbidity and mortality rates than dichorionic twins. Approximately 50% of MC ...

科研

医学

Epidural Intracranial Pressure Measurement in Rats Using a Fiber-optic Pressure Transducer

Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and cardiac arrest. This elevation may result in further neurological injury, in the form of transtentorial herniation1,2,3,4, midbrain compression, neurological deficit or increased cerebral infarct2,4. Current therapies are often inadequate to control elevated ICP in the clinical setting5,6,7 . Thus there is a need for accurate methods of ICP measurement in animal models to further our understanding of the basic mechanisms and to develop new treatments for elevated ICP.
In both the clinical and experimental setting ICP cannot be estimated without direct measurement. Several methods of ICP catheter insertion currently exist. Of these the intraventricular catheter has become the clinical 'gold standard' of ICP measurement in humans8. This method involves the partial removal of skull and the instrumentation of the catheter through brain tissue. Consequently, intraventricular catheters have an infection rate of 6-11%9. For this reason, subdural and epidural cannulations have become the preferred methods in animal models of ischemic injury. 
Various ICP measurement techniques have been adapted for animal models, and of these, fluid-filled telemetry catheters10 and solid state catheters are the most frequently used11,12,13,14,15. The fluid-filled systems are prone to developing air bubbles in the line, resulting in false ICP readings. Solid state probes avoid this problem (Figure 1). An additional problem is fitting catheters under the skull or into the ventricles without causing any brain injury that might alter the experimental outcomes. Therefore, we have developed a method that places an ICP catheter contiguous with the epidural space, but avoids the need to insert it between skull and brain. 
An optic fibre pressure catheter (420LP, SAMBA Sensors, Sweden) was used to measure ICP at the epidural location because the location of the pressure sensor (at the very tip of the catheter) was found to produce a high fidelity ICP signal in this model. There are other manufacturers of similar optic fibre technologies13 that may be used with our methodology. Alternative solid state catheters, which have the pressure sensor located at the side of the catheter tip, would not be appropriate for this model as the signal would be dampened by the presence of the monitoring screw. 
Here, we present a relatively simple and accurate method to measure ICP. This method can be used across a wide range of ICP related animal models.

A novel technique to record the pressures within the skull is described. The minimally invasive method uses a fibre-optic pressure sensing system to accurately measure intracranial pressure (ICP) in anaesthetized rats without causing significant brain trauma. The technique may be used in a wide range of experimental models.

硬膜外颅内压测量中使用的光纤压力传感器的大鼠

Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and ...

A Simple Method of Mouse Lung Intubation

A simple procedure to intubate mice for pulmonary function measurements would have several advantages in longitudinal studies with limited numbers or expensive animal. One of the reasons that this is not done more routinely is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper we demonstrate a procedure that eliminates one of the major hurdles associated with this intubation, that of visualizing the trachea during the entire time of intubation. The approach uses a 0.5 mm fiberoptic light source that serves as an introducer to direct the intubation cannula into the mouse trachea. We show that it is possible to use this procedure to measure lung mechanics in individual mice over a time course of at least several weeks. The technique can be set up with relatively little expense and expertise, and it can be routinely accomplished with relatively little training. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice, thereby minimizing the number of mice needed and increasing the statistical power by using each mouse as its own control.

This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea.

一个简单的方法小鼠肺插管

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

The In ovo CAM-assay as a Xenograft Model for Sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for personalized medicine after stratification, explaining the current interest in developing a reproducible and low-cost xenotransplant model for this disease. The chick chorioallantoic membrane is a natural immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition, it is easily accessed, manipulated and imaged using optical and fluorescence stereomicroscopy. Histology further allows detailed analysis of heterotypic cellular interactions.
This protocol describes in detail the in ovo grafting of the chorioallantoic membrane with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines (Saos-2 and SW1353). The chick survival rates are up to 75%. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior. For localized grafting of single cell suspensions, ECM gel provides significant advantages over inert containment materials. The Ki67 proliferation index is related to the distance of the cells from the surface of the CAM and the duration of application on the CAM, the latter determining a time frame for the addition of therapeutic products.

The In ovo CAM-assay as a Xenograft Model for Sarcoma

The in ovo chorioallantoic membrane (CAM) is grafted with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior.

该<em&gt;在OVO</em&gt; CAM作为一个肉瘤Xenograftmodel的检测

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

颈动脉输液的紫杉烷类化合物在小鼠体内的药代动力学和药效学分析

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

A Simple Critical-sized Femoral Defect Model in Mice

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all limb bone fractures fail to heal completely due to the extent of the trauma, disease, or age of the patient. Our ability to improve bone regenerative strategies is critically dependent on the ability to mimic serious bone trauma in test animals, but the generation and stabilization of large bone lesions is technically challenging. In most cases, serious long bone trauma is mimicked experimentally by establishing a defect that will not naturally heal. This is achieved by complete removal of a bone segment that is larger than 1.5 times the diameter of the bone cross-section. The bone is then stabilized with a metal implant to maintain proper orientation of the fracture edges and allow for mobility. 
Due to their small size and the fragility of their long bones, establishment of such lesions in mice are beyond the capabilities of most research groups. As such, long bone defect models are confined to rats and larger animals. Nevertheless, mice afford significant research advantages in that they can be genetically modified and bred as immune-compromised strains that do not reject human cells and tissue.
Herein, we demonstrate a technique that facilitates the generation of a segmental defect in mouse femora using standard laboratory and veterinary equipment. With practice, fabrication of the fixation device and surgical implantation is feasible for the majority of trained veterinarians and animal research personnel. Using example data, we also provide methodologies for the quantitative analysis of bone healing for the model.

Animal models are frequently employed to mimic serious bone injury in biomedical research. Due to their small size, establishment of stabilized bone lesions in mice are beyond the capabilities of most research groups. Herein, we describe a simple method for establishing and analyzing experimental femoral defects in mice.

一个简单的临界尺寸股骨缺损模型小鼠

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all ...

Utilizing an Orally Dissolving Strip for Pharmacological and Toxicological Studies: A Simple and Humane Alternative to Oral Gavage for Animals

Prior to testing novel therapeutics in humans, short and long term preclinical (i.e., animal), repetitive pharmacological and toxicological testing is required. In most cases, the preferred route of administration is via oral delivery. At the present time, oral delivery is mostly accomplished using an oral gavage procedure, in part, because it can achieve consistent and precise dosing in the animal model. Although this method is well established it does have complications that can result in a high rate of animal attrition. To this end, the procedure introduced here describes an alternative to the oral gavage method in which the desired drug is incorporated into a tastant, orally dissolving strip (ODS) that can simply be presented to the test animal where it is then rapidly taken up with minimal manipulation of the test subject. Herein, we demonstrate that preclinical, oral drug delivery using the ODS method represents a safe, convenient, and humane alternative to oral gavage.

The following protocol describes a novel method for chronic oral drug administration using an orally dissolving strip (ODS) in lieu of the more commonly used oral gavage method. We demonstrate that preclinical, oral drug delivery using the ODS method represents a safe, convenient, and humane alternative to oral gavage.

利用一种口腔溶解地带的药理毒理研究:一个简单的和人道的替代口服灌胃动物

Prior to testing novel therapeutics in humans, short and long term preclinical (i.e., animal), repetitive pharmacological and toxicological testing is ...

An Improved Method for Rapid Intubation of the Trachea in Mice

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity is a commonly used model to study both acute lung injury and fibrosis, and multiple methods have been developed for administering bleomycin (and other toxic agents) into the lungs. However, many of these approaches, such as transtracheal instillation, have inherent drawbacks, including the need for strong anesthetics and survival surgery. This paper reports a quick, reproducible method of intratracheal intubation that involves mild inhaled anesthesia, visualization of the trachea, and the use of a surrogate spirometer to confirm exposure. As a proof of concept, 8-12 week old C57BL/6 mice were administered either 2.0 U/kg of bleomycin or an equivalent volume of PBS, and both damage and fibrotic endpoints were measured post-exposure. This procedure allows researchers to treat a large cohort of mice in a relatively short period with little expense and minimal post-procedure care.

This article presents a rapid and simple method for administering bleomycin directly into the mouse trachea via intubation. Key advantages of this method are that it is highly reproducible, easy to master, and does not require specialized equipment or lengthy recovery times.

在小鼠气管插管快速的改进方法

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity ...

A Method for Quantifying Upper Limb Performance in Daily Life Using Accelerometers

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. It has become important to measure a person's activities in daily life, and not just measure their capacity for activity in the structured environment of a clinic or laboratory. A wearable sensor that is now enabling measurement of daily movement is the accelerometer. Accelerometers are commercially-available devices resembling large wrist watches that can be worn throughout the day. Data from accelerometers can quantify how the limbs are engaged to perform activities in peoples' homes and communities. This report describes a methodology to collect accelerometry data and turn it into clinically-relevant information. First, data are collected by having the participant wear two accelerometers (one on each wrist) for 24 h or longer. The accelerometry data are then downloaded and processed to produce four different variables that describe key aspects of upper limb activity in daily life: hours of use, use ratio, magnitude ratio, and the bilateral magnitude. Density plots can be constructed that visually represent the data from the 24 h wearing period. The variables and their resultant density plots are highly consistent in neurologically-intact, community-dwelling adults. This striking consistency makes them a useful tool for determining if upper limb daily performance is different from normal. This methodology is appropriate for research studies investigating upper limb dysfunction and interventions designed to improve upper limb performance in daily life in people with stroke and other patient populations. Because of its relative simplicity, it may not be long before it is also incorporated in clinical neurorehabilitation practice.

This protocol describes a method to quantify upper limb performance in daily life using wrist-worn accelerometers.

一种使用加速度计量化上肢表现在日常生活的方法

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. ...

Observational Study Protocol for Repeated Clinical Examination and Critical Care Ultrasonography Within the Simple Intensive Care Studies

Longitudinal evaluations of critically ill patients by combinations of clinical examination, biochemical analysis and critical care ultrasonography (CCUS) may detect adverse events of interventions such as fluid overload at an early stage. The Simple Intensive Care Studies (SICS) is a research line that focuses on the prognostic and diagnostic value of combinations of clinical variables.
The SICS-I specifically focused on the use of clinical variables obtained within 24 h of acute admission for prediction of cardiac output (CO) and mortality. Its sequel, SICS-II, focuses on repeated evaluations during ICU admission. The first clinical examination by trained researchers is performed within 3 h after admission consisting of physical examination and educated guessing. The second clinical examination is performed within 24 h after admission and includes physical examination and educated guessing, biochemical analysis and CCUS assessments of heart, lungs, inferior vena cava (IVC) and kidney. This evaluation is repeated at days 3 and 5 after admission. CCUS images are validated by an independent expert, and all data is registered in an online secured database. Follow-up at 90 days includes registration of complications and survival status according to patient&#39;s medical charts and the municipal person registry. The primary focus of SICS-II is the association between venous congestion and organ dysfunction.
The purpose of publishing this protocol is to provide details on the structure and methods of this on-going prospective observational cohort study allowing answering multiple research questions. The design of the data collection of combined clinical examination and CCUS assessments in critically ill patients are explicated. The SICS-II is open for other centers to participate and is open for other research questions that can be answered with our data.

<meta charset="utf8"></head><body>简单重症监护研究中重复临床检查和重症监护超声检查的观察性研究方案

Structured protocols are necessary to provide answers on research questions in critically ill patients. The Simple Intensive Care Studies (SICS) provides an infrastructure for repeated measurements in critically ill patients including clinical examination, biochemical analysis and ultrasonography. SICS projects have specific focus but the structure is flexible to other investigations.

简单重症监护研究中重复临床检查和重症监护超声检查的观察性研究方案

Longitudinal evaluations of critically ill patients by combinations of clinical examination, biochemical analysis and critical care ultrasonography (CCUS) ...