我们感谢博士。黛安·帕尔梅里和NCI的帕特里夏Steeg的为我们提供的MDA-MB231Br细胞。我们感谢拉尔夫·梅森博士，杰森Reneau先生和雷蒙娜·洛佩斯女士为技术和合议支持。这项工作是由美国国防部IDEA奖W81XWH-08-1-0583和W81XWH-12-1-0317支持部分。核磁共振实验是在先进的成像研究中心，一个国家卫生研究院BTRP＃P41-RR02584设施进行，1S10RR02564801根据与VisualSonics VEVO 770进行超声引导下腔内注射。

所有动物的程序批准了得克萨斯大学西南医学中心的机构动物护理和使用委员会。 1。该MDA-MB231/Br-GFP细胞的制备<ol><li>检索和文化MDA-MB231/Br-GFP细胞在含10％FBS，1％谷氨酰胺和1％青霉素/链霉素的DMEM培养基（好心由Drs。帕尔迈和Steeg的，美国国立癌症研究所提供）。 </li><li>观察的细胞和培养基的颜色的情况下，和更换培养基每2-3天。 </li><li>胰蛋白酶消化并收集细胞，当达到80％汇合按照步骤概述1.3.1-1.3.5： <ol><li>彻底清除旧的介质，再加5毫升的PBS（1X）轻轻洗细胞。从盘中取出PBS。 </li><li>添加1.5毫升胰蛋白酶的菜;倾斜的菜轻轻，以确保所有的细胞都受胰蛋白酶。放盘回细胞培养箱，并保持在37℃1分钟。 </li><li>就拿盘开出孵化器，并观察光学显微镜下的细胞，以保证细胞分离从培养皿中。 </li><li>加入3毫升培养基以停止胰蛋白酶的作用。收集在离心管中的细胞混合物。在2000 rpm离心5分钟离心混合物。 </li><li>小心地取出介质和悬浮细胞在5ml无血清培养基中至均匀。 </li></ol></li><li>计数细胞的适当数量和1.75×10 5个细胞在100μl体积的最终浓度重悬它们在无血清的DMEM培养基中。 <ol><li>取50微升的细胞混合，并加入50微升台盼蓝。混匀后，取10微升混合物，并小心地加入到细胞计数幻灯片。使用多个样本，以保证精确的细胞数目的估计。 </li><li>坚守在细胞计数滑动，一端在时间和细胞计数器开始计数自动。取平均的所有点票结果，并计算总细胞数。 </li><li>再次离心细胞，重悬在他们无血清培养基中的适当体积，以导致在1.75×10 5个细胞微升体积中的最终浓度。 </li></ol></li><li>前腔内注射冰上位置细胞。 </li></ol> 2。超声成像引导下腔内注射<ol><li>使用雌性裸鼠（BALB / c小鼠女/女）6-8周龄之间。 </li><li>登陆成像系统。初始化传感器704（40兆赫）。 </li><li>开始一项新的研究，并填写信息。 </li><li>麻醉（3％isoflurane/100％的O 2中的感应腔室），并通过鼻锥的整个过程中保持用异氟烷（2％）的动物在100％O 2（1分米3 /分钟）。当没有退缩反射与脚趾捏观察麻醉确认。 </li><li>设置在成像表的温度提高到37℃。用胶带将麻醉鼠标在仰卧位加热成像表。 </li><li>保持超声波凝胶在37℃之前，即时通讯老化。应用凝胶鼠标的胸部。 </li><li>安装换能器的支架。降低换能器，直到所需的成像深度。移动载物台，直到左心室是确定与升主动脉作为界标（ 图1A）。锁定时，左心室的清晰视图可视化的阶段。 </li><li>吸取100μl的细胞混合物的成1ml注射器，用22号针头。放置和固定注射器的针筒安装。 </li><li>将注射器向前迈进鼠标胸部然后小心地移动一边到另一边，直到针尖在视场成像（进入鼠标之前）。 </li><li>调节针的高度和角度瞄准向下左心室。 </li><li>及时通过皮肤和肌肉层穿透注射器针头插入肋间空间分成超声成像引导下左心室。 指示针的成功插入到左侧脑室CLE是新鲜的动脉血（粉红色对比暗红色静脉血）回流到注射器</li><li>注入的细胞混合物缓慢（ 图1B）。 </li><li>完成后，退出针，抬起传感器，用干净纱布蘸超声凝胶取出磁带。 </li><li>准备一个干净的笼子里有一个预热垫。 </li><li>将动物的垫和观察动物，直到完全康复。 </li><li>监测动物的行为，每24小时为两天。 </li><li>一般条件和实验小鼠并发症的神经系统体征定期监测。 </li></ol> 3。颅内肿瘤发展的MRI监测<ol><li>使用一个9.4特斯拉磁体监测脑转移瘤的颅内发展。 </li><li>两个星期后肿瘤植入发起MRI和重复每周一次长达三个星期。 </li><li>稳重动物用3％异氟醚和维护他们根据基因RAL麻醉（1.5％异氟醚）。 </li><li>监控和​​维护动物的体温和呼吸恒定的整个实验过程。 </li><li>高分辨率的多层（14个切片用1毫米厚，没有间隙）笔1 -和T 2加权冠状图像，覆盖从额叶该区域后颅窝，具有以下参数获得的：T 1加权图像：自旋回波多个切片（SEMS），TR / TE = 400 msec/20毫秒，矩阵：256×256，FOV 20×20毫米，在平面分辨率：78 X 78微米2。 T2加权图像：快速自旋回波多个切片（FSEMS）序列，TR / TE = 2500 msec/48毫秒，8回波串，矩阵：256×256，FOV 20×20毫米，在平面分辨率：78 X 78微米28,9。 肿瘤病灶出现比对T 2加权像正常脑组织明亮。 </li><li>肿瘤的大小是由手工概述enhan对T 2加权像上确定通过使用由我们所撰写8 MATLAB程序的每个图像上CING质量的部分。 鉴于大多数肿瘤直径小于切片厚度（1毫米）小的，肿瘤的大小被表示为在平面区域，而不是音量。 </li></ol> 4。 H＆E染色确认的转移<ol><li>后最后MR平扫牺牲小鼠，解剖荷瘤大脑和嵌入华侨城中，在冷冻-80℃ </li><li>第一个系列的10微米厚的冠状脑标本与低温恒温器。 </li><li>对脑切片进行H＆E染色。 </li></ol>

<ol>
	<li>Schouten, L. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence+of+brain+metastases+in+a+cohort+of+patients+with+carcinoma+of+the+breast,+colon,+kidney,+and+lung+and+melanoma.">Incidence of brain metastases in a cohort of patients with carcinoma of the breast, colon, kidney, and lung and melanoma.</a> Cancer. 94, 2698-2705 (2002).</li><li>Lin, N. U., 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=CNS+metastases+in+breast+cancer.">CNS metastases in breast cancer.</a> J. Clin. Oncol. 22, 3608-3617 (2004).</li><li>Eichler, A. 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没有利益冲突的声明。

在本研究中，我们已经证明，超声成像引导下左心室注射保证了精度，使每一个动物在此开发研究脑转移，无动物死亡进行了观察。影像引导下注入的妙处在于皮肤，最后进入左心室的针入度的路径可以被监控，并根据图像，这是从手动过程，需要严格的解剖标志遵循不同的调整。 脑转移瘤的颅内发展，目前的认识主要是基于对动物模型4,10组织学研究。然而，组织?...

脑转移瘤是成人最常见的颅内恶性肿瘤。预后极差，用4-6个月，即使积极治疗，中位生存期。乳腺癌脑转移1-3的高发病率的三大主要癌症之一。几个脑嗜乳腺癌线能够心内或颈动脉注射后4开发脑转移。直接注射肿瘤细胞进入左心室可以绕过肺毛细血管床，从而增加形成的脑转移，同时尽量减少内脏转移的发病率。在MDA-MB231Br线是使用最广泛的人类乳腺癌行开发脑转移在啮齿动物模型5,6中的一个。 像许多其他的研究4,7，我们已经在我们以前的研究进行心内注射的手动过程。然而，用手动获得只有50％的成功率注射小鼠的一小部分来自重复侵入性操作，如果死之前失败的试验。这里，我们引入了利用成像引导程序，以确保在注射脑寻乳腺癌细胞到无胸腺小鼠的左心室。纵向高分辨率MRI应用于遵循脑转移瘤的颅内发展。

<table border="0" cellpadding="0" cellspacing="0" width="783">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">DMEM</td>
			<td style="width:136px;">HyClone</td>
			<td style="width:87px;">SH30022.01</td>
			<td style="width:147px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Trypsin</td>
			<td style="width:136px;">Mediatech</td>
			<td style="width:87px;">25-051-C1</td>
			<td style="width:147px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Automatic cell counter</td>
			<td style="width:136px;">Biorad</td>
			<td style="width:87px;">TC10</td>
			<td style="width:147px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Isoflurane</td>
			<td style="width:136px;">Baxter International Inc.</td>
			<td style="width:87px;">1001936060</td>
			<td style="width:147px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">VisualSonics Vevo 770 High-Resolution Imaging System </td>
			<td>Visual Sonics Inc.</td>
			<td></td>
			<td style="width:147px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">9.4T horizontal bore magnet with a Varian INOVA Unity system </td>
			<td>Agilent</td>
			<td style="width:87px;"> </td>
			<td style="width:147px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">O.C.T Compound</td>
			<td>Sakura Finetek USA</td>
			<td style="width:87px;">4583</td>
			<td style="width:147px;"> </td>
		</tr>
	</tbody>
</table>

ultrasound imaging-guided intracardiac injection to develop a mouse model of breast cancer brain metastases followed by longitudinal mri

乳腺癌脑转移，在IV期乳腺癌患者的30％的发生，是与高死亡率有关。中位生存期仅为6个月。关键是要具有合适的动物模型来模拟在临床情况中转移细胞的血液动力学扩散。在这里，我们介绍使用小动物超声成像引导一个准确的注射脑热带乳腺癌细胞到无胸腺裸鼠的左心室。纵向磁共振成像是用来评估颅内启动和脑转移瘤的生长。超声引导下腔内注射不仅确保了精确的注射，并在此较高的成功率也显著降低死亡率，比我们以前的手动过程。 体内高分辨率MRI高信号允许多灶性病变的可视化，小到310微米直径对T 3周注射后2加权图像。福卢W-MRI起来发现颅内肿瘤的生长和分布在整个脑转移的数量增加。

用核磁共振（在图面的分辨率78微米）的高空间分辨率，高信号病变可以被确定为小，中直径310微米（ 图2）。因为在这项研究中的转移是非常小的，坏死和水肿的发展是最小的，对T 2加权图像上的高信号病变真正表示的肿瘤块。 纵向MRI研究允许在肿瘤生长的活体无创评估。 如图3所示 ，高分辨率的MRI却能3周后，心内注射（ <stro...

Watch this Scientific Journal Video about Ultrasound Imaging-guided Intracardiac Injection to Develop a Mouse Model of Breast Cancer Brain Metastases Followed by Longitudinal MRI at JoVE.com

Ultrasound Imaging-guided Intracardiac Injection to Develop a Mouse Model of Breast Cancer Brain Metastases Followed by Longitudinal MRI

乳腺癌脑转移瘤小鼠模型的建立与超声成像引导下腔内注射MDA-MB231/Br-GFP细胞。多灶性脑转移的发展已经采用高分辨率9.4ŤMRI检查被监控纵。

超声成像引导下腔内注射到患乳腺癌脑转移其次是纵向的MRI的小鼠模型

Breast cancer brain metastasis, occurring in 30% of breast cancer patients at stage IV, is associated with high mortality. The median survival is only 6 ...

ultrasound-imaging-guided-intracardiac-injection-to-develop-mouse

Research

JoVE Journal

Medicine

18.6K 次观看.  University of Texas Southwestern Medical Center. 乳腺癌脑转移瘤小鼠模型的建立与超声成像引导下腔内注射MDA-MB231/Br-GFP细胞。多灶性脑转移的发展已经采用高分辨率9.4ŤMRI检查被监控纵。 

视频: Ultrasound Imaging-guided Intracardiac Injection to Develop a Mouse Model of Breast Cancer Brain Metastases Followed by Longitudinal MRI

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

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

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

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

在小鼠模型体内生物发光成像乳腺癌脑转移的肿瘤乏氧动力学

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

科研

医学

MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a significant obstacle to pharmaceutical treatments of brain disorders as it limits the passage of molecules from the vasculature into the brain tissue to molecules less than approximately 500 Da in size6. FUS induced BBB disruption (BBBD) is temporary and reversible4 and has an advantage over chemical means of inducing BBBD by being highly localized. FUS induced BBBD provides a means for investigating the effects of a wide range of therapeutic agents on the brain, which would not otherwise be deliverable to the tissue in sufficient concentration. While a wide range of ultrasound parameters have proven successful at disrupting the BBB2,5,7, there are several critical steps in the experimental procedure to ensure successful disruption with accurate targeting. This protocol outlines how to achieve MRI-guided FUS induced BBBD in a rat model, with a focus on the critical animal preparation and microbubble handling steps of the experiment.

Microbubble-mediated focused ultrasound disruption of the blood-brain barrier (BBB) is a promising technique for non-invasive targeted drug delivery in the brain1-3. This protocol outlines the experimental procedure for MRI-guided transcranial BBB disruption in a rat model.

MRI引导下的破坏血脑屏障在大鼠模型经颅聚焦超声

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a ...

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

利用磁共振成像，容积电脑断层扫描及超音波的裸鼠乳腺癌骨转移大鼠模型血管生成的多模态成像

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

实时成像在乳腺癌小鼠模型中的肿瘤微环境对药物的反应

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

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 ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

乳腺癌细胞侵袭定量使用三维（3D）模型

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

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 ...

A Preclinical Murine Model of Hepatic Metastases

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, murine pancreatic tumor model of hepatic metastases via a hemispleen injection of syngeneic murine pancreatic tumor cells is described. This model mimics many of the clinical conditions in patients with metastatic disease to the liver. Mice consistently develop metastases in the liver allowing for investigation of the metastatic process, experimental therapy testing, and tumor immunology research.

A preclinical, murine model of hepatic metastases performed via a hemispleen injection technique.

肝转移的临床前小鼠模型

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

注射原位乳腺癌细胞到小鼠乳腺脂肪垫来研究肿瘤的生长。

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

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

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

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

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