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细胞培养液中的未补充有胎牛血清。使用微波炉（850瓦，持续40秒，然后3×15秒;挑起每个微波会话之间的解决方案），煮沸解决方案，并以完全溶解琼脂糖。高压灭菌20分钟，这1％的琼脂糖溶液，在120℃，以确保免缝。利蒂注 ：小心高压釜协议之前完全溶解琼脂糖。不均匀性可能会危及球体的形成。 </li><li>一旦从高压釜中取出，倒10毫升在100×20毫米培养皿中的1％琼脂糖溶液。离开培养皿20分钟，在室温下，使1％的琼脂糖溶液凝固。 </li><li>加琼脂糖高于10毫升的PBS中，以保持湿度。通过将它们包装用封口膜，以避免蒸发密封的琼脂糖包被培养皿的盖子。培养皿可以在4℃可保存长达2个月，如果适当地由PBS层加湿。 </li></ol></li><li> 球体文化 <ol><li>在一个75 厘米的烧瓶中，培养的神经胶质瘤细胞在其推荐的介质作为一个单层。 </li><li>从1％琼脂糖包被培养皿中加入1.5ml“预处理中的”从含胶质瘤细胞的二维单层瓶收集请更换PBS秒。 </li><li>从含有神经胶质瘤细胞的2D单层烧瓶中取出培养基。 </li><li>轻轻地冲洗该单层用10毫升PBS中。 </li><li>加入2毫升的胰蛋白酶/ EDTA溶液0.05％和孵育在37℃ 注烧瓶4分钟：该孵育时间可根据所使用的细胞株不同而不同。 </li><li>加入8毫升培养介质停止胰蛋白酶的作用下，轻轻冲洗细胞悬液注意 ：小心不要冲洗空气中的细胞悬液，因为它会增加细胞死亡。 </li><li>放6毫升的细胞悬液在无菌小瓶中。 </li><li>离心小瓶4分钟800转。 </li><li>去除上清，轻轻悬浮细胞沉淀在3毫升培养液中。 </li><li>种子细胞悬浮液中制备的琼脂糖包被的培养皿。 </li><li>将培养皿于37℃在5％CO 2的气氛中进行2〜3天，直到所希望的直径的球状体形成。 </li></ol></li></oL&gt; 2，球体和植入窗<ol><li> 该喷射系统的下游 <ol><li>干净的玻璃毛细管（直径1毫米），通过超声处理在70％乙醇中10分钟。漂洗两次，水配售前的孵化器内直至干燥。 </li><li>为了准备一个小的股票拉几个清洗玻璃毛细管用吸管拉马。 </li><li> 300-350微米的典型的外部直径和250-300微米的导管：通过刮擦末端上的一块薄纸，得到所需尺寸的斜面末端打破拉毛细管的尖端。在此成形过程中，控制用粗视其眼配备有刻度的米拉毛细管的直径。 </li><li>毛细管的非斜切末端连接到一个3路歧管使用的是一块塑料管，其内径适合的毛细管的外径。 </li><li>使用塑料tubinG，连接歧管的另外两种方式的 25微升微升注射器和一个1毫升注射器装入直方兼容的矿物油。 </li><li>调整歧管选择建立微升注射器和1ml注射器之间的通道。清除 该微升注射器的活塞和落后的注入矿物油进入微升注射器，直到漏出通过推动1ml注射器的活塞。更换微升注射器的活塞，同时注意不要让气泡在管。 </li><li>调整歧管选择建立毛细管和1ml注射器之间的通道。填充毛细管用矿物油，直到它从前端漏出，同时小心不要离开气泡在管中。 </li><li>调整所述歧管选择建立微升注射器和毛细管之间的连接。驱逐约10微升矿物油。 </li><li>浸毛细管尖端插入PBS溶液和使用微升注射器的规格，吸在PBS中的毛细管各5μl。请注意，油和PBS之间的弯月面清晰可见。 </li><li>修复毛细血管上一个3轴显微操作下手术粗视配件。此系统将被用于毛细管目标视觉控制下注射部位。 </li></ol></li><li> 球体植入 <ol><li>轻轻吸入异氟醚（在1〜1.5分钟1.5％，在空气中）稳重的鼠标感应室。 </li><li>麻醉动物用腹膜内注射氯胺酮/赛拉嗪（120毫克/千克，12毫克/千克）的混合物组成。需要注意的是麻醉往往降低了动物的体温。我们建议进行手术的房间在26°C和保持动物的体温与底层thermocontrolled加热垫。 </li><li>适用眼膏，避免眼睛干燥。 </li><li>剃除动物的头皮。 </li><li>地方动物在使用耳棒和吹嘴立体定位框架。 </li><li>清洁用聚维酮碘（3％的皂，然后用10％的溶液）的皮肤。 </li><li>制成1厘米长的切口沿纵向在用手术刀头皮的中间。用剪刀剪和去除上面的顶骨皮肤。 </li><li>用手术刀刀片轻轻取出头骨上方的骨膜。慷慨地适用于骨生成一个粗糙表面后水泥坚持顶氰基丙烯酸酯。 </li><li>钻下一个手术粗视顶骨以产生直径4mm的开颅手术。慷慨地添加冰冷的PBS整个程序，以防止加热过程中含青霉素（1,000单位/毫升）和Streptomicin（1毫克/毫升）。用镊子和湿无菌纱布取出小碎骨。小心钻至少相距1mm顶颅骨缝，以避免出血。 </li><li>瘦骨在那里的玻璃窗将被密封在开颅手术的边界。目标是到ENsure的后面的平面与大脑和玻璃之间的最大接触面玻璃窗的定位。慢慢地用钳子取出骨头和清洁暴露硬脑膜，脑膜与PBS溶液。 </li><li>取直径5mm的圆形玻璃盖玻片，用酒精清洁，然后用纸巾擦干。尝试使用盖玻片作为盖的开颅手术，并确认大脑的大平面进入与玻璃接触。如有必要取出盖玻片，进一步薄边的骨头。继续进行，直到大脑可以被挤压扁，如果轻轻施加压力，盖玻片一次到位。 </li><li>再次用酒精清洁盖玻片，用纸巾擦干，并保存它拆开。 </li><li>用26号针头使硬脑膜，母校在开颅尚未避开主要血管的中心孔。请确保不要损坏脑实质作为这一步的目的只是打开硬脑膜，脑膜。 </li><li>轻轻擦拭DURA-脑膜无线TH PBS溶液以去除出血性血液。顶盖采用了一块纸巾，同时准备球体注入。 </li><li>在步骤2.1准备的球体喷射系统，吸圆的球体从步骤1.2准备的培养皿毛细管内径（约200-250微米）拟合。球体应该同来，大约5微升培养基。根据它的重量应该倒下的毛细作用的一角。 </li><li>除去用于支付的开颅手术，并根据喷射系统定位动物的湿纸巾。 </li><li>降低注射针，直到它触及的持续时间母校作出的洞。然后，通过250微米再次降低它。等待30秒。 </li><li>慢慢用微升注射器的活塞（2-3微升），而泵出多余的液体，薄薄的一张纸巾注入球体。等待30秒，然后由50微米轻轻提起注射针向表面。再等待30秒，repea通过步长为50微米吨的提升过程，直到表面。等待时间避免了提取，将粘在吸管的球体。 </li><li>使用荧光型宏观确认的球状体的大脑中的存在。 </li><li>拌100-300微米直径的交联葡聚糖凝胶珠在培养皿中的PBS溶液。等待1分钟和一些鱼水合珠。将它们放在相邻的开颅手术骨。 </li><li>在宏观调控中，选择一个小珠与类似硬脑膜，脑膜开口的大小的直径。切使用镊子两半的交联葡聚糖凝胶珠。 </li><li>轻轻把交联葡聚糖凝胶半珠与朝向旋转椭圆体的凸面的喷射孔。注意不要移除球体，并轻轻按下向下，直到凹的前沿进入与周围硬脑膜，脑膜接触。 </li><li>把氰基丙烯酸酯胶在载玻片上滴;沾了牙签的尖端进入德罗p来取少量胶水。通过围绕冲压胶迅速密封该交联葡聚糖凝胶半珠的边缘到相邻的硬脑膜，脑膜。小心以避免胶的交联葡聚糖凝胶半珠的牙签进行快速和精确的移动。避免多余的胶水，可以传播和阻碍，如果不能阻止成像一旦干燥。等待2分钟的胶水干后清洁开颅手术和周围骨用PBS液。 </li></ol></li><li> 植入窗 <ol><li>把直径5 mm的圆形盖玻片开颅以上（见2.2.12）。盖玻片的边缘必须在减薄的头骨在开颅手术的外部。盖玻片必须与硬脑膜-脑膜和薄骨上的边缘相接触。这是非常重要的是要有硬脑膜-脑膜和盖玻片之间的直接接触，否则疤痕组织可能发展，阻碍光学清晰度。 </li><li>用纸巾，吸取PBS在thË盖玻片边缘，使溶液涂敷在盖玻片中心的小的压力时，不会完全覆盖开颅手术。这将确保骨，玻璃和大脑上的感兴趣区域的侧面胶合在一起。 </li><li>保持在盖玻片用钳子中心的一个小的压力，同时轻轻施加氰基丙烯酸酯在骨和盖玻片之间的边界。胶水会自发地传播，直到用PBS液的接口。溶液将作为给定的胶的疏水性的屏障。密封应该是有效的，在不到一分钟的时间，但举一个极端的小心不要移动盖玻片，直到它被固定。这将会产生的持续时间母校从而导致手术的失败氰基丙烯酸酯泄漏。 </li><li>万一胶水将涂布在玻璃板的上方，通过做螺旋升降用微型手术刀刀片除去它，因为它可能会以其他方式减少的光学清晰度。小心不要弄破玻璃盖玻片超级碗克因压力过大的程序。 </li><li>通过施加牙科用粘固剂在朝向相邻的颅骨盖玻片的边缘巩固玻璃固定。确保覆盖整个颅骨外露，直到头皮。使用额外的水泥，建立侧壁周围的盖玻片以创建所需的目标浸泡池。该牙科用粘固剂将固化在少于10分钟。 </li></ol></li></ol> 3，术后护理和成像准备<ol><li> 术后护理 <ol><li>从立体定位框架中取出鼠标，注入Dexamethazon（0.2毫克/千克）和Rimadyl（5毫克/公斤），SC在骨盆区域，奠定了她在笼子里有一个温暖的组织巢穴。 </li><li>监测动物，直到麻醉的效果已经消失。在手术后一般2〜3小时，老鼠是完全移动。确保动物有一个易于获得食物和水。含葡萄糖琼脂（提供动物琼脂糖3％，葡萄糖3％）。 </li><li>每天监测动物的体重和注入Dexamethazon（0.2毫克/千克）和Rimadyl（5毫克/千克）SC为第一10d的手术后。对于道德方面的考虑，安乐死的小鼠，当损失超过其原来重量的15％。颅内压力增加与肿瘤的大小，导致运动障碍的动物。这是肿瘤细胞系依赖的，并且发生在各种延迟手术后。特别应注意以表征实验中使用的细胞系的端点。 </li></ol></li><li> 成像准备 <ol><li>轻轻吸入异氟醚镇静的小鼠中的感应腔（在空气中进行1〜1.5分钟，1.5％）。 </li><li>麻醉动物用腹膜内注射氯胺酮/赛拉嗪（100毫克/千克，10毫克/千克）的混合物组成。这种麻醉通常允许观察45分钟。要执行较长的成像会议，鼠标被放置在在持续吸入异氟醚空气（0.25-0.75％）。 </li><li>适用眼膏，避免眼睛干燥。 </li><li>把动物的立体定位框架和阻止颅骨与earbars。 </li><li>来可视化血管，荧光染料可被静脉内注射。 </li><li>如果该窗口出现脏，它可以通过轻轻地除去碎片用薄刀片和一个组织的角落进行清洁。不要用酒精清洁窗口，因为它并不总是与牙科用粘固剂相容，它可以冷却脑的窗口的下面。 </li></ol></li></ol>

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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=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. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+12.">Ch. 12.</a> CNS Cancer, Cancer Drug Discovery and Development. , 227-241 (2009).</li><li>Ulrich, T. A., de Juan Pardo, E. M., Kumar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanical+rigidity+of+the+extracellular+matrix+regulates+the+structure,+motility,+and+proliferation+of+glioma+cells.">The mechanical rigidity of the extracellular matrix regulates the structure, motility, and proliferation of glioma cells.</a> Cancer Res. 69, 4167-4174 (2009).</li></ol>

作者宣称，他们有没有竞争的财务权益。

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

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。

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-orthotopic-glioblastoma-mouse-model-maintaining-brain-parenchymal

Research

JoVE Journal

Medicine

19.6K 次观看.  Aix Marseille University. 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。 

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

An Orthotopic Model of Murine Bladder Cancer

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and subsequent cell adhesion. After their bladders are transurethrally catheterized and drained, animals are again catheterized to permit insertion of a platinum wire into bladders without damaging the urethra or bladder. The catheters are made of Teflon to serve as an insulator for the wire, which will conduct electrical current into the bladder to create a burn injury. An electrocautery unit is used to deliver 2.5W to the exposed end of the wire, burning away extracellular layers and providing attachment sites for carcinoma cells that are delivered in suspension to the bladder through a subsequent catheterization. Cells remain in the bladder for 90 minutes, after which the catheters are removed and the bladders allowed to drain naturally. The development of tumor is monitored via ultrasound. Specific attention is paid to the catheterization technique in the accompanying video.

Bladder tumors are established in female mice in a minimally invasive fashion through catheterization, local cauterization, and subsequent adhesion of carcinoma cells to the burn sites.

一个小鼠膀胱癌原位模型

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and ...

科研

医学

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

多光子成像在口腔鳞状细胞癌原位小鼠模型的肿瘤细胞的侵袭

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model

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

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

在原位小鼠脑肿瘤模型结合放疗

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

An Orthotopic Mouse Model of Anaplastic Thyroid Carcinoma

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer cells into immunodeficient mice. Subcutaneous xenograft models have been valuable for preclinical screening and evaluation of new therapeutic treatments. There are a number of advantages to using a subcutaneous model; 1) rapid, 2) reproducible, and 3) tumor establishment, growth, and response to therapeutic agents may be monitored by visual inspection. However, substantial evidence has shed light on the short-comings of subcutaneous xenograft models1-3. For instance, medicinal treatments demonstrating curative properties in subcutaneous xenograft models often have no notable impact on the human disease. The microenvironment of the site of xenographic transplantation or injection lies at the heart of this dissimilarity.
Orthotopic tumor xenograft models provide a more biologically relevant context in which to study the disease. The advantages of implanting diseased cells or tissue into their anatomical origin equivalent within a host animal includes a suitable site for tumor-host interactions, development of disease-related metastases and the ability to examine site-specific influence on investigational therapeutic remedies. Therefore, orthotopic xenograft models harbor far more clinical value because they closely reproduce human disease. For these reasons, a number of groups have taken advantage of an orthotopic thyroid cancer model as a research tool4-7.
Here, we describe an approach that establishes an orthotopic model for the study of anaplastic thyroid carcinoma (ATC), which is highly invasive, resists treatment, and is virtually fatal in all diagnosed patients. Cultured ATC cells are prepared as a dissociated cellular suspension in a solution containing a basement membrane matrix. A small volume is slowly injected into the right thyroid gland. Overall appearance and health of the mice are monitored to ensure minimal post-operative complications and to gauge pathological penetrance of the cancer. Mice are sacrificed at 4 weeks, and tissue is collected for histological analysis. Animals may be taken at later time-points to examine more advance progression of the disease. Production of this orthotopic mouse model establishes a platform that accomplishes two objectives: 1) further our understanding of ATC pathology, and 2) screen current and future therapeutic agents for efficacy in combating ATC.

Generation of an orthotopic mouse model of anaplastic thyroid carcinoma is described here. This technique employs surgical placement of human anaplastic thyroid cancer cells into the thyroid of immunodeficient mice, thus creating a more clinically relevant setting to study disease progression as well as screen innovative therapeutic interventions.

甲状腺未分化癌的原位小鼠模型

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer ...

An Orthotopic Bladder Cancer Model for Gene Delivery Studies

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are needed. The tumor microenvironment can significantly influence tumor development and therapy response. It is therefore often desirable to grow tumor cells in the organ from which they originated. This protocol describes an orthotopic model of bladder cancer, in which MB49 murine bladder carcinoma cells are instilled into the bladder via catheterization. Successful tumor cell implantation in this model requires disruption of the protective glycosaminoglycan layer, which can be accomplished by physical or chemical means. In our protocol the bladder is treated with trypsin prior to cell instillation. Catheterization of the bladder can also be used to deliver therapeutics once the tumors are established. This protocol describes the delivery of an adenoviral construct that expresses a luciferase reporter gene. While our protocol has been optimized for short-term studies and focuses on gene delivery, the methodology of mouse bladder catheterization has broad applications.

Implantation of cancer cells into the organ of origin can serve as a useful preclinical model to evaluate novel therapies. MB49 bladder carcinoma cells can be grown within the bladder following intravesical instillation. This protocol demonstrates catheterization of the mouse bladder for the purpose of tumor implantation and adenoviral delivery.

原位膀胱癌模型的基因传递研究

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

人类前列腺癌转移的原位小鼠模型

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no effective treatment for it yet. Limitations of currently used animal models impede understanding of the pathology mechanism. Multiphoton microscopy allows studying cells and tissues within intact animal brains longitudinally under physiological and pathological conditions. Here, we describe two models of acute brain injury studied by means of two-photon imaging of brain cell behavior under posttraumatic conditions. A selected brain region is injured with a sharp needle to produce a trauma of a controlled width and depth in the brain parenchyma. Our method uses stereotaxic prick with a syringe needle, which can be combined with simultaneous drug application. We propose that this method can be used as an advanced tool to study cellular mechanisms of pathophysiological consequences of acute trauma in mammalian brain in vivo. In this video, we combine acute brain injury with two preparations: cranial window and skull thinning. We also discuss advantages and limitations of both preparations for multisession imaging of brain regeneration after trauma.

Acute brain trauma is a severe injury that has no adequate treatment to date. Multiphoton microscopy allows studying longitudinally the process of acute brain trauma development and probing therapeutical strategies in rodents. Two models of acute brain trauma studied with in vivo two-photon imaging of brain are demonstrated in this protocol.

急性脑外伤小鼠其次是纵向双光子成像

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no ...

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 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 ...

An Orthotopic Mouse Model of Spontaneous Breast Cancer Metastasis

Metastasis is the primary cause of mortality of breast cancer patients. The mechanism underlying cancer cell metastasis, including breast cancer metastasis, is largely unknown and is a focus in cancer research. Various breast cancer spontaneous metastasis mouse models have been established. Here, we report a simplified procedure to establish orthotopic transplanted breast cancer primary tumor and resultant spontaneous metastasis that mimic human breast cancer metastasis. Combined with the bioluminescence live tumor imaging, this mouse model allows tumor growth and progression kinetics to be monitored and quantified. In this model, a low dose (1 x 104 cells) of 4T1-Luc breast cancer cells was injected into BALB/c mouse mammary fat pad using a tuberculin syringe. Mice were injected with luciferin and imaged at various time points using a bioluminescent imaging system. When the primary tumors grew to the size limit as in the IACUC-approved protocol (approximately 30 days), mice were anesthetized under constant flow of 2% isoflurane and oxygen. The tumor area was sterilized with 70% ethanol. The mouse skin around the tumor was excised to expose the tumor which was removed with a pair of sterile scissors. Removal of the primary tumor extends the survival of the 4T-1 tumor-bearing mice for one month. The mice were then repeatedly imaged for metastatic tumor spreading to distant organs. Therapeutic agents can be administered to suppress tumor metastasis at this point. This model is simple and yet sensitive in quantifying breast cancer cell growth in the primary site and progression kinetics to distant organs, and thus is an excellent model for studying breast cancer growth and progression, and for testing anti-metastasis therapeutic and immunotherapeutic agents in vivo.

An orthotopic breast cancer primary tumor model and surgical removal of primary tumor to extend mouse life to generate spontaneous metastasis are described. The tumor growth and progression are monitored and quantified by luciferase fluorescence imaging.

自发性乳腺癌转移的原位小鼠模型

Metastasis is the primary cause of mortality of breast cancer patients. The mechanism underlying cancer cell metastasis, including breast cancer ...

Intravital Microscopy and Thrombus Induction in the Earlobe of a Hairless Mouse

Thrombotic complications of vascular diseases are one leading cause of morbidity and mortality in industrial nations. Due to the complex interactions between cellular and non-cellular blood components during thrombus formation, reliable studies of the physiology and pathophysiology of thrombosis can only be performed in vivo. Therefore, this article presents an ear model in hairless mice and focuses on the in vivo analysis of microcirculation, thrombus formation, and thrombus evolution. By using intravital fluorescence microscopy and the intravenous (iv) application of the respective fluorescent dyes, a repetitive analysis of microcirculation in the auricle can easily be performed, without the need for surgical preparation. Furthermore, this model can be adapted for in vivo studies of different issues, including wound healing, reperfusion injury, or angiogenesis. In summary, the ear of hairless mice is an ideal model for the in vivo study of skin microcirculation in physiological or pathophysiological conditions and for the evaluation of its reaction to different systemic or topical treatments.

The ear model of the hairless SKH1-Hrhr mouse enables intravital fluorescence microscopy of microcirculation and phototoxic thrombus induction without prior surgical preparation in the examined microvascular bed. Therefore, the ear of the hairless mouse is an excellent in vivo model to study the complex interactions during microvascular thrombus formation, thrombus evolution, and thrombolysis.

活体显微镜及血栓诱导在无毛小鼠的耳垂

Thrombotic complications of vascular diseases are one leading cause of morbidity and mortality in industrial nations. Due to the complex interactions ...