Name: an orthotopic glioblastoma mouse model maintaining brain parenchymal physical constraints and suitable for intravital two-photon microscopy
Uploaded: 2014-04-21T15:15:00
Description: Watch this Scientific Journal Video about An Orthotopic Glioblastoma Mouse Model Maintaining Brain Parenchymal Physical Constraints and Suitable for Intravital Two-photon Microscopy at JoVE.com

作者衷心感谢KK Fenrich博士，MC博士。 Amoureux酒店，体育韦伯和A. Jaouen有益的讨论; M.侯赛因·，C.梅尼尔，M. Metwaly，S. Bensemmane，J. Bonnardel，在IBDML动物设施和PicSIL成像平台IBDML的技术支持工作人员的工作人员。这项工作得到了补助国立研究所杜癌症（INCA-DGOS-INSERM6038）遗传资源，法新社国立德拉RECHERCHE（ANR JCJC PathoVisu3Dyn），联合会的支持倒拉RECHERCHE河畔乐Cerveau（FRC）以FD，由国际足球联合会奖学金德拉RECHERCHEMédicale和CancéropolePACA到CR。

所有试验程序都按照法国法律，并遵守欧洲共同体理事会指令11月24日，1986（86/609/EEC）实验动物的护理和使用进行。对动物的研究被授权方向DépartementaleDES服务Vétérinaires德罗讷河口省（许可证D-13-055-21），并经普罗旺斯蔚蓝海岸编号为14（项目87-04122012）的伦理委员会。 1，制备球状体<ol><li> 的琼脂糖包被培养皿的准备 <ol><li>混合1克琼脂糖用100ml细胞培养液中的...

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D., 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=Phase+III+randomized+study+of+radiotherapy+plus+procarbazine,+lomustine,+and+vincristine+with+or+without+BUdR+for+treatment+of+anaplastic+astrocytoma:+final+report+of+RTOG+9404.">Phase III randomized study of radiotherapy plus procarbazine, lomustine, and vincristine with or without BUdR for treatment of anaplastic astrocytoma: final report of RTOG 9404.</a> Int J Radiat Oncol Biol Phys. 58, 1147-1152 (2004).</li><li>Piao, Y., 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=Glioblastoma+resistance+to+anti-VEGF+therapy+is+associated+with+myeloid+cell+infiltration,+stem+cell+accumulation,+and+a+mesenchymal+phenotype.">Glioblastoma resistance to anti-VEGF therapy is associated with myeloid cell infiltration, stem cell accumulation, and a mesenchymal phenotype.</a> Neuro Oncol. 14, 1379-1392 (2012).</li><li>Zhai, H., Heppner, F. L., Tsirka, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia/macrophages+promote+glioma+progression.">Microglia/macrophages promote glioma progression.</a> Glia. 59, 472-485 (2011).</li><li>Studwell, A. J., Kotton, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+shift+from+cell+cultures+to+creatures:+in+vivo+imaging+of+small+animals+in+experimental+regenerative+medicine.">A shift from cell cultures to creatures: in vivo imaging of small animals in experimental regenerative medicine.</a> Mol Ther. 19, 1933-1941 (2011).</li><li>Ustione, A., Piston, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+introduction+to+multiphoton+microscopy.">A simple introduction to multiphoton microscopy.</a> J Microsc. 243, 221-226 (2011).</li><li>Ricard, 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=Short-term+effects+of+synchrotron+irradiation+on+vasculature+and+tissue+in+healthy+mouse+brain.">Short-term effects of synchrotron irradiation on vasculature and tissue in healthy mouse brain.</a> J Synchrotron Radiat. 16, 477-483 (2009).</li><li>von Baumgarten, L., 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=Bevacizumab+has+differential+and+dose-dependent+effects+on+glioma+blood+vessels+and+tumor+cells.">Bevacizumab has differential and dose-dependent effects on glioma blood vessels and tumor cells.</a> Clin Cancer Res. 17, 6192-6205 (2011).</li><li>Helmchen, F., Denk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+tissue+two-photon+microscopy.">Deep tissue two-photon microscopy.</a> Nat Methods. 2, 932-940 (2005).</li><li>Mostany, R., Portera-Cailliau, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+craniotomy+surgery+procedure+for+chronic+brain+imaging.">A craniotomy surgery procedure for chronic brain imaging.</a> J Vis Exp. , 680 (2008).</li><li>Lathia, J. D., 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=Direct+in+vivo+evidence+for+tumor+propagation+by+glioblastoma+cancer+stem+cells.">Direct in vivo evidence for tumor propagation by glioblastoma cancer stem cells.</a> PLoS One. 6, (2011).</li><li>Madden, K. S., Zettel, M. L., Majewska, A. K., Brown, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+tumor+imaging:+live+imaging+of+glioma+by+two-photon+microscopy.">Brain tumor imaging: live imaging of glioma by two-photon microscopy.</a> Cold Spring Harb Protoc. 2013, (2013).</li><li>Winkler, F., 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=Imaging+glioma+cell+invasion+in+vivo+reveals+mechanisms+of+dissemination+and+peritumoral+angiogenesis.">Imaging glioma cell invasion in vivo reveals mechanisms of dissemination and peritumoral angiogenesis.</a> Glia. 57, 1306-1315 (2009).</li><li>Newcomb, E., Zagzag, D., Van Meir, E. 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这种方法允许使用光学成像的方法来监测过几天和几周的原位植入胶质瘤的生长。同一动物可以随后的病理过程中经受几乎所有的大脑成像模态;但在双光子显微镜具体的准备提供了独特的机会来实现生活的动物的大脑中的亚细胞分辨率。我们的协议提出的优点来执行的肿瘤生长进入脑实质，而不是额外的durally因为它发生如神经胶质瘤细胞可通过受损硬脑膜离开中枢神经系统。在不存在由大脑环?...

胶质母细胞瘤表现为脑肿瘤在成人与12个月的中位生存期最积极的形式和5％的5年存活率。临床管理依赖于手术，放疗和化疗常常结合使用。然而，这些治疗的效果仍然姑息1-3。 截至目前，大多数神经肿瘤学的研究都依赖于那些只能提供静态视图和对荷瘤动物的大同伙进行牺牲在不同的时间点（例如，见4,5）技术。随访方法基于活体成像的最新发展使得研究脑胶质瘤生长和肿瘤细胞以及它们对同一动物随着时间的推移病理生理微环境之间的相互作用。这开辟了道路，以独家的资料片，这是迄今无法实现的6。转基因动物表达荧光标记的目标细胞可使用d，来研究在本文中肿瘤细胞和例如神经元之间的特异性相互作用。 在过去的十年中，活体双光子显微镜7已经成为基本的神经肿瘤学研究和临床前试验8,9为其进行深活体观察小鼠大脑能力的黄金标准（&gt; 500微米以下的持续时间母校）与一个微米级的空间分辨率10。用活体双光子显微镜用植入慢性颅窗11 orthotopical动物模型中，也能够按照肿瘤进展时间的推移在同一小鼠9,12。 一这些先前公布的动物模型中的主要缺点是但它们不模仿支配肿瘤生长的硬脑膜，脑膜未注射的细胞悬浮液9,13,14后密封的物理约束。胶质瘤细胞可能泄漏的硬膜外空间转化原位胶质瘤模型成异位之一。 这里介绍的动物模型包括在注射荧光胶质瘤细胞在大脑皮质在200μm的深度接着硬脑膜，脑膜用交联葡聚糖凝胶半珠和直方兼容胶的密封的旋转椭球体的。然后将肿瘤生长被限制在脑实质，它维护的病理生理物理约束。上述肿瘤植入一种慢性玻璃窗允许活体双光子显微镜一件容易的光纤接入。利用转基因动物表达荧光标记的目标细胞有可能随着时间的推移来进行后续的脑胶质瘤生长，并研究其相互作用及其微环境（这里用神经元和血管突出了荧光葡聚糖）。

<table border="0" cellpadding="0" cellspacing="0" width="1337">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="38">
			<td height="38" style="height:38px;width:425px;">Drill</td>
			<td style="width:383px;">Dremel (Germany)</td>
			<td style="width:156px;">398</td>
			<td style="width:375px;">any high quality surgical bone drill would suffice</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:425px;">Drill burr (#1/4 Carbide Round Burr)</td>
			<td style="width:383px;">World Precision Instruments (USA)</td>
			<td>501860 (#1/4)</td>
			<td>also sold by Harvard Apparatus</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Tissue scissors</td>
			<td style="width:383px;">World Precision Instruments (USA)</td>
			<td>14395</td>
			<td style="width:375px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Dumont tweezers M5S</td>
			<td style="width:383px;">World Precision Instruments (USA)</td>
			<td>501764</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Dental cement</td>
			<td>GACD (USA)</td>
			<td>12-565 &#38; 12-568</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Cyanoacrylate</td>
			<td>Eleco-EFD (France)</td>
			<td>Cyanolit 201</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Glass capillaries without filament</td>
			<td>Clark Electromedical Instruments (UK)</td>
			<td>GC100-15</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Microliter syringe (25 &#181;l)</td>
			<td>Hamilton (USA)</td>
			<td>702</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Micromanipulator</td>
			<td style="width:383px;">World Precision Instruments (USA)</td>
			<td>Kite-R</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">T derivation (3-way stopcock - Luer lock)</td>
			<td style="width:383px;">World Precision Instruments (USA)</td>
			<td>14035-10</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Stereotactic frame (mouse adaptor)</td>
			<td style="width:383px;">World Precision Instruments (USA)</td>
			<td>502063</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Glass coverslips</td>
			<td>Warner Instruments (USA)</td>
			<td>CS-5R (64-0700)</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Cross-linked dextran gel (Sephadex) G50 Coarse 100-300 &#181;m beads</td>
			<td>Available from various suppliers including Sigma (Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Eye ointment</td>
			<td>TVM (France)</td>
			<td>Ocry-gel</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Fluorescence macroscope</td>
			<td>Leica MZFLIII (Germany)</td>
			<td></td>
			<td>also sold by other companies</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Two-photon microscope</td>
			<td>Zeiss LSM 7MP (Germany)</td>
			<td></td>
			<td>also sold by other companies (Nikon, &#8230;)</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Infrared tunable femtosecond laser (Ma&#239;-Ta&#239;)</td>
			<td>Spectra Physics (USA)</td>
			<td></td>
			<td>also sold by other companies</td>
		</tr>
	</tbody>
</table>

an orthotopic glioblastoma mouse model maintaining brain parenchymal physical constraints and suitable for intravital two-photon microscopy

胶质母细胞瘤（GBM）是脑肿瘤可至今没有根治的治疗的最积极的形式。 这种病理小鼠模型依赖于注射神经胶质瘤细胞的悬浮液进入脑实质以下硬脑膜-母校的切​​口。而细胞必须被表面注入到可访问的活体双光子显微镜，浅表注射失败复述的病理生理条件。事实上，通过注射道逃逸大多数肿瘤细胞到达外硬膜空间，他们不正常的扩张速度快于没有从实质机械约束。 我们的改进灶性植入胶质瘤球体而不是注射神经胶质瘤细胞中的悬浮液中的大脑皮质的表面层，而且在通过交联葡聚糖凝胶半珠被粘在surroun堵塞注射部位不仅包括鼎实质和密封，以DURA-母校与氰基丙烯酸酯。总之，这些措施的执行的生理扩张脑实质内的肿瘤细胞的浸润。开颅手术终于关上了玻璃窗凝成的头骨，使慢性成像在周中没有瘢痕组织的发展。 利用接枝我们已经表明，神经胶质瘤细胞，神经元（ 例如，大鼠Thy1-CFP小鼠）和血管（通过静脉内注射一种荧光染料的突出显示）之间发生相互作用的动力学可以通过活体可视化的荧光肿瘤细胞的荧光的转基因动物的该疾病的进展过程中双光子显微镜。 的可能性，像一个肿瘤在一个最低限度的脑损害环境的微观决议代表当前GBM动物模型应该有利于神经肿瘤学和药物测试领域的改进。 </p&gt;

一旦外科协议来执行（ 图1），动物可通过荧光显微镜的方法观察到数周，直到牺牲。炎症反应可能有一个或两个星期内消失，手术后进行观察。肿瘤的生长可通过各种显微技术包括荧光宏观和双光子显微镜（ 图2）被观察到。这里所示的示例图像，实现了对荧光型宏观和双光子显微镜耦合到飞秒脉冲红外可调谐激光器和家用修改，以允许下面的20X水浸物镜（1.0NA）动物的...

Watch this Scientific Journal Video about An Orthotopic Glioblastoma Mouse Model Maintaining Brain Parenchymal Physical Constraints and Suitable for Intravital Two-photon Microscopy at JoVE.com

An Orthotopic Glioblastoma Mouse Model Maintaining Brain Parenchymal Physical Constraints and Suitable for Intravital 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

我们已经建立了原位皮质胶质母细胞瘤模型小鼠的活体双光子显微镜的概括了生物物理的限制，通常在作怪肿瘤的生长过程。一种慢性玻璃窗替换上面的肿瘤颅骨使后续的肿瘤进展时间的推移由双光子显微镜。

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