我们非常感谢博士罗伯特·S·Kerbel（多伦多大学，新宁研究所，加拿大多伦多，加拿大）的技术帮助和专业知识在此过程中发展的实验室。我们还要感谢桑德拉·塞克斯顿博士和罗斯威尔公园癌症研究所部实验动物资源。这项工作是从罗斯威尔公园联盟基金会（以JMLE）裁决的支持。

1。原位肾肿瘤植入<ol><li>细胞培养<ol><li>在此之前原位移植，种植鼠标RENCA LUC细胞单层至75％汇合。 </li><li>以下胰​​蛋白酶消化并重新悬浮于含5％FBS的培养基，离心分离细胞，以1,000 rpm，4℃，5分钟，重复3次，在PBS中洗涤。然后悬浮细胞在无血清培养基，以5×10 4 RENCA LUC / 5μL无血清培养基中的浓度。 注：根据不同的变种或使用细胞系，细胞可以以1:1的比例基质胶和无血清培养基的放缓植入后高攻击小鼠细胞的生长和传播的再悬浮。 </li></ol></li><li>手术和细胞移植 注：其中所描述的所有动物的研究，包括实验终点的维护和决心，根据机构动物护理和使用委员会（IACUC）P分别进行rotocol批准罗斯威尔公园癌症研究所。 <ol><li>麻醉用异氟醚（2-3％）1 BALB / c小鼠。捏脚和检查反射，以确保有足够的麻醉。适用于兽医软膏的眼睛，以防止干燥。将鼠标右侧卧，刮胡子前和后肢之间左侧。用酒精和碘酒沿背腰部区域为无菌切口准备区。 </li><li>用手术剪启动1厘米的皮肤切口，在最后肋和髋关节之间的纵向方向。使用钝性分离剪刀皮肤下松开结缔组织。用手术剪，以0.5厘米的切口在长度方向上在腹壁。用弯钳轻轻向下推周围开放性伤口 - 这将exteriorize肾和注射前允许器官的温柔固定。 </li><li>加载汉密尔顿注射器5μL准备好的细胞混合物。对于子囊IMPLANtations，2注入的方法可以采用： <ol><li>浅表性：与斜面边缘向上平行延伸到肾囊下的纵定向肾，插入针（但薄壁段）。一旦针到位，注入所有的细胞，直到白色小泡的形式。慢慢地从胶囊取出针头，并立即涂抹在注射用无菌棉签吸收任何泄漏和防止细胞传播。 </li><li>内部：与斜面边缘并从肾脏相反的一侧开始到最后的植入位置，通过肾脏的内部插入针直至针是可见的（但不打孔）亚荚膜空间。注入的所有单元格，直到白色泡沫形式和拭注射部位，以防止任何泄漏。 </li></ol></li><li>轻轻肾脏返回到体腔中。关闭用5-0可吸收缝线薇乔腹部体壁。一旦完成，拉扯皮肤层一起放置2-3胜OUND剪辑。确保剪辑是坚定和间隔均匀，以确保没有创面暴露。在回收前施用500微升0.9％的NaCl和100μl丁丙诺啡（0.01毫克/毫升）的皮下用25号针头。定期监测夹子安置和稳定，并在10天后删除。 </li></ol></li></ol> 2，肾切除术/原发肿瘤去除<ol><li>遵循相同的协议，用于鼠标麻醉，固定，切口，肾暴露在章节1.2.1和1.2.2（前述）。 </li><li>使用第二对镊子轻轻除去从尾端连接的脂肪组织和来自肾脏的颅端肾上腺分离肾脏。同时轻轻抓住肾脏，用5-0可吸收缝线薇乔使输尿管周围一个双结，肾动脉，静脉。上面结慢慢的安全，然后切成取出肾脏。如果马上失血，或有任何迹象是保持足够的风险在缝合输尿管，动脉，静脉的，然后用cauterizer，而不是剪刀剪下上面结。 </li><li>一旦肾脏被删除，请仔细检查是否有出血的动脉栓并在必要时使用cauterizer。关闭用5-0薇乔腹部体壁。拉扯皮肤层一起放置2-3伤口剪辑如1.2.4中所述的相同的注意事项。每日密切监视动物手术后痛苦的迹象，出血或活动受限。按照动物的组织指导方针安全。 </li></ol> 3，监测转移进程和本地化的端点<ol><li>转移性疾病定量：发光法监测<ol><li>看到以前在JOVE描述监测转移使用的细胞转染荧光素酶和利用精诺真IVIS成像系统11,12量化方法。 </li></ol></li><li>视觉剖检指南转移性分布的比较评估化<ol><li>根据使用的端点，包括窘迫征象，呼吸困难，体重减轻等机构的指导方针牺牲动物 注：密切监测动物是至关重要的，因为自发的转移进程可以是充满变数和快速。 </li><li>用手术剪刀开始一直延续了胸腔，以下颌尿道口上方的切口。用钝端剪刀将皮肤从腹壁的肌肉分开。 </li><li>开始沿手臂和腿部切口。剥离的皮肤了。仔细检查皮肤筋膜和腹壁的结节，那么，浅表淋巴结肿大：腹股沟，臂丛，腋和颈浅。 </li><li>做一个切口在腹腔壁和沿到膜片上的左，右的侧面切割。有一个横向切去除腹壁的前部裸露的内脏。 </li><li>检查内脏f的外观原状或极显着的肿瘤。还要注意胃和肠道中的内容;如果它们是空的，鼠标可能不会已经能够吃。如果内容是黑暗的，这可能表明上/下消化道出血的证据。 </li><li>目测评分各器官肿瘤的存在或不存在下，分配一个总的分数，各小鼠组（每组4只动物被用作例如，在图1D-ⅰ）。 注释：1）淋巴结肿大并不一定意味着它是一个肿瘤生长;如果是很大的规模，很结实，发白的颜色，它可能是一个转移灶。 2）过度乳白色或混浊的液体在腹部指示腹水。腹水可能包括固体堆积在腹部脏器，特别是肝脏和胃的叶之间。 3）对器官如肺，多发性结节可以存在并计数为动物个体之间的比较（参见第13为例）。 </li><li>通过小心地从一个喷瓶取出并用PBS肝，脾，肾和胰腺：确定总的肿瘤个体腹部器官或异常。 </li><li>去除腹部器官后，检查腹腔淋巴结，包括腰，骶，肾和肠系膜淋巴结。 </li><li>评估隔膜的转移扩散的证据。 注：在膜片广泛的传播可能会导致它失去收缩能力，导致或吃力呼吸损失。 </li><li>去掉膜片，使两个横向切口于胸腔，除去前面的部分，以暴露肺和心脏。 </li><li>轻轻抓住并提起食道和切割高于心脏气管取出的肺和心脏。评估组织的结节。挤压心脏。缺乏弹性的一致性可能预示肿瘤的存在。 </li><li>用手术剪透过颅骨线性切割。轻轻地取出小块头盖骨，直到t他整个大脑被暴露。看看整个大脑可见结节或异常变色。 </li></ol></li></ol>

<ol>
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作者什么都没有透露。

此协议的目的是使用同基因肿瘤小鼠模型来描述植入/切除技术来评估临床相关的自发转移性疾病。目前，大多数临床前研究，评估新的实验疗法不包括转移性疾病的研究中，只有少数概括的原发肿瘤生长，手术切除，并最终自发转移扩散的阶段。迄今为止，基因工程小鼠模型（GEMMS）产生的诱发自发性肾细胞疾病进展（见14摘要）不包括后期转移和外科干预策略迄今尚未见报道。因此，自...

死亡的患者的肾细胞癌（RCC）的主要的原因是，通常发生在手术切除原发肿瘤在肾生长的全身性转移疾病。然而，很少有临床前肿瘤模型评价实验治疗的小鼠包括转移性疾病，少仍然忠实地复述局部增长，手术和自发微转移的发生和发展1-3的临床分期。在测试这个差距，已成为新的治疗方法，因为有时出现在动物模型中引人注目的抗肿瘤作用并不总是翻译成同样成功治疗的患者4的评价越来越重要。在结果这种差异可能源于局部的异位或原位原发性肿瘤模型和晚期转移性疾病的5-7之间的差的药物疗效。在碾压的情况下，只有少数研究采用建立了nimal协议，其中包括自发复发的疾病，谁通常有荷瘤肾脏模仿患者全部或部分除去2,3。在小鼠模型中测试这种情况的原因缺乏变化。首先，有高动物成本和肿瘤细胞的选择和转移潜能的固有可变性。例如，人肾细胞系往往很少转移，并且必须在多个回合的主原位植入和转移性选择派生的变种，始终传播并形成远处病灶进行选择（见这样的人细胞系的推导8-10的说明） 。相反，小鼠细胞免疫功能的模型往往表现积极，低细胞数量必须与基质胶，以减少直接的全身播散3注入。其次，在执行适当的植入，手术切除（切除术），和跟踪技术困难（和量化）自发metastati℃生长可以是具有挑战性和几个关键变量采用这种技术时（见讨论的细节）需要考虑。本协议的目的是描述原位移植，切除（切除术），并监测自发转移性肾细胞癌的疾病的基本步骤（以及潜在的缺陷），并提供一个指引，规范（以及更广泛的）科学实验室中使用该评估实​​验治疗的疗效。

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		<tr height="21">
			<td height="21" style="height:21px;width:397px;">DMEM-high glucose with Pyr. And L-Glutamine</td>
			<td style="width:147px;">Corning</td>
			<td style="width:119px;">10-013-CV</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">FBS</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:119px;">10437-028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">0.25% Trypsin EDTA</td>
			<td style="width:147px;">Corning</td>
			<td style="width:119px;">25-053-CL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">1x DPBS without Calcium &#38; Magnesium</td>
			<td style="width:147px;">Corning</td>
			<td style="width:119px;">21-031-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Matrigel</td>
			<td style="width:147px;">BD Biosciences</td>
			<td style="width:119px;">354234</td>
			<td>must be kept on ice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Artifical tears-lubricant opthalmic ointment</td>
			<td style="width:147px;">Akorn Animal Health</td>
			<td style="width:119px;">17478-162-35</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Pocket pro pet trimmer</td>
			<td style="width:147px;">Braintree scientific</td>
			<td style="width:119px;">CLP9931B</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:397px;">Alcohol swab</td>
			<td style="width:147px;">VWR</td>
			<td style="width:119px;">326895</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Betadine solution swab</td>
			<td style="width:147px;">VWR</td>
			<td style="width:119px;">67618-152-01</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;">MICRO DISSECTING sissors straight,blunt - 25mm blades - 4.5&#34;&#160;</td>
			<td style="width:147px;">Southpointe surgical</td>
			<td style="width:119px;">RS-5982</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Iris Forceps, serrated, curved, 10cm long</td>
			<td style="width:147px;">Kent scientific</td>
			<td style="width:119px;">INS15915</td>
			<td>need two of these</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:397px;">10&#181;l Hamilton syringe</td>
			<td style="width:147px;">Hamilton</td>
			<td style="width:119px;">7635-01</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:397px;">30G, 45 degree, RN needle</td>
			<td style="width:147px;">Hamilton</td>
			<td style="width:119px;">7803-07</td>
			<td></td>
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			<td height="24" style="height:24px;width:397px;">Sterile cotton tipped appicator</td>
			<td style="width:147px;">VWR</td>
			<td style="width:119px;">10805-144</td>
			<td></td>
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			<td height="21" style="height:21px;width:397px;">High temperature cautery kit</td>
			<td style="width:147px;">Kent scientific</td>
			<td style="width:119px;">INS500392</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:397px;">5-0 coated Vicryl, conventional cutting needle&#160;</td>
			<td style="width:147px;">Ethicon</td>
			<td style="width:119px;">J834</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Reflex clip applier for 7mm clips</td>
			<td style="width:147px;">Kent scientific</td>
			<td style="width:119px;">INS500343</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Reflex clips, 7mm, non-sterile</td>
			<td style="width:147px;">Kent scientific</td>
			<td style="width:119px;">INS500344</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Removing forceps, 12cm lone</td>
			<td style="width:147px;">Kent scientific</td>
			<td style="width:119px;">INS500347</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">0.9% Sodium Chloride</td>
			<td style="width:147px;">Baxter Healthcare</td>
			<td style="width:119px;">2B1322</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Buprenorphine 0.01mg/mL</td>
			<td style="width:147px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">25G 5/8&#34; needle</td>
			<td style="width:147px;">VWR</td>
			<td style="width:119px;">BD305122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">1mL syringe w/out needle</td>
			<td style="width:147px;">VWR</td>
			<td style="width:119px;">BD309659</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">D-Luciferin</td>
			<td style="width:147px;">Gold Bio technology</td>
			<td style="width:119px;">LUCK-1G</td>
			<td></td>
		</tr>
	</tbody>
</table>

modeling spontaneous metastatic renal cell carcinoma (mrcc) in mice following nephrectomy

其中一个关键的挑战，新的实验治疗肾细胞癌（RCC）提高了测试的模型，忠实地复述早期和晚期转移性疾病进展的发展。典型肿瘤植入模型利用异位或原位原发性肿瘤植入，但很少有全身性的自发转移性疾病，模仿临床上。本协议描述的关键步骤，制定农村信用社疾病进展阶段类似患者。首先，它采用了高转移性小鼠肿瘤细胞系中的同源模型，以显示原位肿瘤细胞植入。方法包括浅表和内部植入与细胞基底膜结合，防止漏电和早期传播子荚膜空间。接着，它描述了程序的荷瘤肾（肾切除术）切除术，具有重要的前和手术后的小鼠护理。最后，它概述了监测和评估的必要步骤微观和宏观转移性疾病的进展，包括生物发光成像，以及提供详细的视觉剖检指南进球全身性疾病的分布。此协议的描述的目的是为了便于普遍使用的临床相关的转移性肾细胞癌模型，以提高未来的治疗试验的预测值。

图1A显示的原理，概述在本协议中总结的详细步骤。有几个重要的因素必须考虑每一步。例如，在步骤1中也示出了两种方法用于子囊肿瘤细胞植入到肾。肿瘤细胞可以被注入到子荚膜空间与一个白色的小泡，确认细胞的渗漏防止通过小心去除针和抽汲过量逸出流体（ 图1B-ⅰ）的局部位置。为了防止泄漏，细胞可植入内部先到达子荚膜空间（ 图1B-ii）中，并?...

Watch this Scientific Journal Video about Modeling Spontaneous Metastatic Renal Cell Carcinoma mRCC in Mice Following Nephrectomy at JoVE.com

Modeling Spontaneous Metastatic Renal Cell Carcinoma mRCC in Mice Following Nephrectomy

可用于临床相关设置评估治疗自发性转移性肾细胞癌（RCC）疾病进展的模型。该协议显示不同的程序原位肾肿瘤细胞植入，适当性肾切除术，最后概述了视觉和生物发光的负担转移和本地化得分尸检指南。

在小鼠模型自发转移性肾细胞癌（MRCC）肾切除

One of the key challenges to improved testing of new experimental therapeutics in renal cell carcinoma (RCC) is the development of models that faithfully ...

modeling-spontaneous-metastatic-renal-cell-carcinoma-mrcc-mice

Research

JoVE Journal

Medicine

Modeling Spontaneous Metastatic Renal Cell Carcinoma (mRCC) in Mice Following Nephrectomy

16.4K 次观看.  Roswell Park Cancer Institute. 可用于临床相关设置评估治疗自发性转移性肾细胞癌（RCC）疾病进展的模型。该协议显示不同的程序原位肾肿瘤细胞植入，适当性肾切除术，最后概述了视觉和生物发光的负担转移和本地化得分尸检指南。 

视频: Modeling Spontaneous Metastatic Renal Cell Carcinoma mRCC in Mice Following Nephrectomy

Single Port Donor Nephrectomy

In 2007, Rane presented the first single port nephrectomy for a small non-functioning kidney at the World Congress of Endourology. Since that time, the use of single port surgery for nephrectomy has expanded to include donor nephrectomy. Over the next two years the technique was adopted for many others types of nephrectomies to include donor nephrectomy. We present our technique for single port donor nephrectomy using the Gelpoint device. We have successfully performed this surgery in over 100 patients and add this experience to our experience of over 1000 laparoscopic nephrectomies. With the proper equipment and technique, single port donor nephrectomy can be performed safely and effectively in the majority of live donors. We have found that our operative times and most importantly our transplant outcomes have not changed significantly with the adoption of the single port donor nephrectomy. We believe that single port donor nephrectomy represents a step forward in the care of living donors.

Single port laparoscopic surgery is changing the standard of care in surgical care like nothing since the laparoscopic technique was introduced 20 years ago. We present out technique of single port donor nephrectomy using the Gelpoint device. We have successfully performed this surgery in 100 patients.

单端口捐赠者肾切除术

In 2007, Rane presented the first single port nephrectomy for a small non-functioning kidney at the World Congress of Endourology.  Since that time, the ...

科研

医学

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats.
Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans.
In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.

Filamentous occlusion of the Middle cerebral artery is a common model for studying ischemic stroke in mice.

建模在小鼠体内的行程 - 中东与灯丝模型大脑中动脉闭塞

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are ...

Mammary Transplantation of Stromal Cells and Carcinoma Cells in C57BL/6J Mice

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular through transplantation of stromal cells and epithelial cells in the mammary gland of mice. Current transplantation models often involve the use of immunocompromised mice due to the different genetic backgrounds of stromal cells and epithelial cells. Extracellular matrices are often used to embed the two different cell types for consistent cell-cell interactions, but involve the use of Matrigel or rat tail collagen, which are immunogenic substrates. The lack of functional T cells from immunocompromised mice prevents accurate assessment of stromal cells on mammary tumor progression in vivo, with important implications on drug development and efficacy. Moreover, immunocompromised mice are costly, hard to breed and require special care conditions. To overcome these obstacles, we have developed an approach to orthotopically transplant stromal cell and epithelial cells into mice from the same genetic background to induce consistent tumor formation. This system involves harvesting normal, carcinoma associated fibroblasts, PyVmT mammary carcinoma cells and collagen from donor C57BL/6J mice. The cells are then embedded in collagen and transplanted in the inguinal mammary glands of female C57BL/6J mice. Transplantation of PyVmT cells alone form palpable tumors 30-40 days post transplantation. Endpoint analysis at 60 days indicates that co-transplantation with fibroblasts enhances mammary tumor growth compared to PyVmT cells transplanted alone. While cells and matrix from C57BL/6J mice were used in these studies, the isolation of cells and matrix and transplantation approach may be applied towards mice from different genetic backgrounds demonstrating versatility. In summary, this system may be used to investigate molecular interactions between stromal cells and epithelial cells, and overcomes critical limitations in immunocompromised mouse models.

In this report, we demonstrate a system to isolate and culture donor cells from the mouse mammary gland, and orthotopically transplant these cells in recipient mice to analyze stromal: epithelial interactions during mammary tumor development.

C57BL/6J小鼠乳腺间质细胞和癌细胞的移植

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular ...

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 ...

Modeling Intracerebral Hemorrhage in Mice: Injection of Autologous Blood or Bacterial Collagenase

Spontaneous intracerebral hemorrhage (ICH) defines a potentially life-threatening neurological malady that accounts for 10-15% of all stroke-related hospitalizations and for which no effective treatments are available to date1,2. Because of the heterogeneity of ICH in humans, various preclinical models are needed to thoroughly explore prospective therapeutic strategies3. Experimental ICH is commonly induced in rodents by intraparenchymal injection of either autologous blood or bacterial collagenase4. The appropriate model is selected based on the pathophysiology of hemorrhage induction and injury progression. The blood injection model mimics a rapidly progressing hemorrhage. Alternatively, bacterial collagenase enzymatically disrupts the basal lamina of brain capillaries, causing an active bleed that generally evolves over several hours5. Resultant perihematomal edema and neurofunctional deficits can be quantified from both models. In this study, we described and evaluated a modified double injection model of autologous whole blood6 as well as an ICH injection model of bacterial collagenase7, both of which target the basal ganglia (corpus striatum) of male CD-1 mice. We assessed neurofunctional deficits and brain edema at 24 and 72 hr after ICH induction. Intrastriatal injection of autologous blood (30 &mu;l) or bacterial collagenase (0.075U) caused reproducible neurofunctional deficits in mice and significantly increased brain edema at 24 and 72 hr after surgery (p&lt;0.05). In conclusion, both models yield consistent hemorrhagic infarcts and represent basic methods for preclinical ICH research.

Clinically relevant animal models of intracerebral hemorrhage (ICH) are needed to extend our knowledge of hemorrhagic stroke and to examine novel therapeutic strategies. In this study, we describe and evaluate two ICH models that implement unilateral injections of either autologous whole blood or bacterial collagenase into the basal ganglia (corpus striatum) of mice.

在小鼠的脑出血模型：注射自体血液或细菌胶原酶

Spontaneous intracerebral hemorrhage (ICH) defines a potentially life-threatening neurological malady that accounts for 10-15% of all stroke-related ...

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

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

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

皮下及肝内人肝癌裸鼠移植瘤的免疫缺陷小鼠的产生

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

Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice:  A Model for Immunotherapy Development

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy and/or cytotoxic chemotherapy. To induce bladder cancer, C57BL/6 mice (MUC1.Tg and wild type) were treated orally with the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN) at 3.0 mg/day, 5 days/week for 12 weeks. To assess the effects of OH-BBN on serum cytokine profile during tumor development, whole blood was collected via submandibular bleeds prior to treatment and every four weeks. In addition, a MUC1-targeted peptide vaccine and placebo were administered to groups of mice weekly for eight weeks. Multiplex fluorometric microbead immunoanalyses of serum cytokines during tumor development and following vaccination were performed. At termination, interferon gamma (IFN-&#947;)/interleukin-4 (IL-4) ELISpot analysis for MUC1 specific T-cell immune response and histopathological evaluations of tumor type and grade were performed. The results showed that: (1) the incidence of bladder cancer in both MUC1.Tg and wild type mice was 67%; (2) transitional cell carcinomas (TCC) developed at a 2:1 ratio compared to squamous cell carcinomas (SCC); (3) inflammatory cytokines increased with time during tumor development; and (4) administration of the peptide vaccine induces a Th1-polarized serum cytokine profile and a MUC1 specific T-cell response. All tumors in MUC1.Tg mice were positive for MUC1 expression, and half of all tumors in MUC1.Tg and wild type mice were invasive. In conclusion, using a team approach through the coordination of the efforts of pharmacologists, immunologists, pathologists and molecular biologists, we have developed an immune intact transgenic mouse model of bladder cancer that expresses hMUC1.

An N-butyl-N-(4-hydroxybutyl)nitrosamine-induced bladder cancer model was developed in human mucin 1 (MUC1) transgenic mice for the purpose of testing MUC1-directed immunotherapy. After administering a MUC1-targeted peptide vaccine, a cytotoxic T lymphocyte response to MUC1 was confirmed by measuring serum cytokine levels and T-cell specific activity.

浸润性膀胱移行细胞癌中的完整的人MUC1转基因小鼠免疫诱导免疫治疗发展模型

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

乳腺癌的一种新的转基因小鼠模型：转移性乳腺癌通过瞄准导管上皮与腺病毒Cre重组酶的启动

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

Modeling Stroke in Mice: Permanent Coagulation of the Distal Middle Cerebral Artery

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical options are available for a small proportion of stroke patients in the acute phase. Current research is intensively searching for novel therapeutic strategies and is increasingly focusing on the sub-acute and chronic phase after stroke because more patients might be eligible for therapeutic interventions in a prolonged time window. These delayed mechanisms include important pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity and regeneration. In order to analyze these mechanisms and to subsequently evaluate novel drug targets, experimental stroke models with clinical relevance, low mortality and high reproducibility are sought after. Moreover, mice are the smallest mammals in which a focal stroke lesion can be induced and for which a broad spectrum of transgenic models are available. Therefore, we describe here the mouse model of transcranial, permanent coagulation of the middle cerebral artery via electrocoagulation distal of the lenticulostriatal arteries, the so-called &#8220;coagulation model&#8221;. The resulting infarct in this model is located mainly in the cortex; the relative infarct volume in relation to brain size corresponds to the majority of human strokes. Moreover, the model fulfills the above-mentioned criteria of reproducibility and low mortality. In this video we demonstrate the surgical methods of stroke induction in the &#8220;coagulation model&#8221; and report histological and functional analysis tools.

Various murine models of middle cerebral artery occlusion (MCAO) are widely used in experimental brain research. Here, we demonstrate the model of transcranial permanent distal MCAO which produces consistent cortical infarction of a size corresponding to damage imposed by the majority of human ischemic strokes.

造型中风小鼠：在远端大脑中动脉永久性混凝

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical ...

Modeling Chemotherapy Resistant Leukemia In Vitro

It is well established that the bone marrow microenvironment provides a unique site of sanctuary for hematopoietic diseases that both initiate and progress in this site. The model presented in the current report utilizes human primary bone marrow stromal cells and osteoblasts as two representative cell types from the marrow niche that influence tumor cell phenotype. The in vitro co-culture conditions described for human leukemic cells with these primary niche components support the generation of a chemoresistant subpopulation of tumor cells that can be efficiently recovered from culture for analysis by diverse techniques. A strict feeding schedule to prevent nutrient fluxes followed by gel type 10 cross-linked dextran (G10) particles recovery of the population of tumor cells that have migrated beneath the adherent bone marrow stromal cells (BMSC) or osteoblasts (OB) generating a "phase dim" (PD) population of tumor cells, provides a consistent source of purified therapy resistant leukemic cells. This clinically relevant population of tumor cells can be evaluated by standard methods to investigate apoptotic, metabolic, and cell cycle regulatory pathways as well as providing a more rigorous target in which to test novel therapeutic strategies prior to pre-clinical investigations targeted at minimal residual disease.

Modeling Chemotherapy Resistant Leukemia In Vitro

The current report summarizes a protocol that can be utilized to model the influence of the bone marrow microenvironment niche on leukemic cells with emphasis placed on enrichment of the most chemoresistant subpopulation.