这项工作得到了 nih R15 1R15DK106666-01A1 (对 j. 刘) 和 nih RO1 HL071556 (J.I. 夏皮罗) 的支持。

所有动物护理和实验均由马歇尔大学机构动物护理和使用委员会 (IACUC) 根据国家卫生研究院 (NIH) 关于实验室动物的护理和使用指南获得批准。雄性 C57BL/6 小鼠 (10-12 周大) 被安置在一个无病原体的动物设施中, 在指定的房间里装有可在12小时的光照/暗循环下提供纯净空气的网箱。食物和水被提供了 ad 随意 . 
 1. 手术准备 注意: 外科手术器械和材料是从不同的来源获得的, 不特定于手术操作。其他来源的仪器和材料也可用于同一目的。有关手术器械的列表, 请参阅 材料目录 . 
<ol> <li> 手术准备 <ol> <li> 在操作表的范围内放置以下内容: 暖垫、卤素灯、氧气罐和异氟醚罐、异氟醚蒸发器、理发剪、脱毛膏、鳞片、灭菌操作套装, 消毒的手术亚麻布, 棉签, 70% 酒精, polyhydroxydine 溶液 (含1% 碘), 0.9% 氯化钠溶液, 1 毫升注射器和针头 (30 克), 3-0 和4-0 丝缝线, 丁丙诺啡和青霉素, 抗生素软膏,润滑油眼膏. </li>
		<li> 清洁一个笼子, 并在床下放置一个热垫, 供小鼠在手术后居住. </li>
	</ol> </li> </ol> 2。一步手术: 左肾极结扎术 注意: 在手术期间保持无菌状态。用无菌垫覆盖蒸压的外科器械。在手术中, 多套外科手术器械, 进行多项手术, 以防止交叉污染。如果需要再次使用仪器, 在多项手术的情况下, 用控告溶液和70% 乙醇对仪器进行消毒, 然后在器官玻璃微珠消毒器中消毒5分钟, 用70% 乙醇消毒操作区域。穿上长袍、面罩 (盖住鼻子和嘴巴)、帽 (盖头) 和一副无菌手套。每次手术后更换手套. 
<ol> <li> 清洗和消毒双手, 并佩戴无菌手术手套。戴头帽和面具. </li>
	<li> 用70% 酒精消毒桌子。将润滑剂眼膏涂抹在老鼠的双眼上, 防止麻醉时眼部干燥. </li>
	<li> 将鼠标放置在异氟醚蒸发器系统的感应室中, 并以2.5% 异氟醚100% 氧 (0.8-1.2 升/分) 的混合物诱导麻醉, 用于 1-2 min. 
	注: 麻醉的功效是由捏尾引起的疼痛反射, 以及眼睑没有眼睑反射而决定的. </li>
	<li> 注射青霉素 (4万单位/千克体重, IM) 和丁丙诺啡 (0.02 毫克/千克体重, SC), 以防止感染和疼痛的手术过程中. </li>
	<li> 将鼠标放在暖垫 (最初设置在42和 #176; c) 上, 用直肠温度计来保持和监测37和 #176 的体温; 在手术过程中。在手术过程中, 将小鼠的鼻子放入鼻锥, 用 0.8-1.2% 的异氟醚与100% 氧 (0.8-1.2 升/分) 的混合物进行通气. </li>
	<li> 将鼠标放在右侧。用理发剪刮掉手术区的毛发, 然后用脱毛膏除去。用 polyhydroxydine 溶液 (含1% 碘) 消毒现场, 然后用70% 乙醇。将蒸压的外科亚麻布对准手术区. </li>
	<li> 做一个〜1.0 厘米腰椎切口和分离的肌肉和筋膜暴露左肾使用手术刀和钳 ( 图 1a ). </li>
	<li> 通过用小钝钳拉动肾脂肪轻轻拉出左肾。在这个过程中, 为了不扰乱肾上腺, 用镊子将肾上腺从肾脏向上移. </li>
	<li> 使用钝钳, 轻轻地从肾脏的上端将结缔组织和肾上腺从肾的上部向肾的中间的幽门处, 血管进入和出口。识别和隔离输尿管. 
	注: 输尿管位于肾脏的下半部, 在结缔组织内, 在光下清晰可见. </li>
	<li> 轻轻地抓住结缔组织并从肾脏组织中分离, 以确保输尿管没有结扎. </li>
	<li> 在极性结扎之前, 使用无菌尺来鉴别肾脏的1/3 部分的优劣。老鼠肾脏通常是大约 1.25-1.5 cm 长. </li>
	<li> 结扎使用3-0 丝线缝合线和适当的力量, 大约0.4 厘米从上级部分往幽门的中部和相似地, 0.4 cm 从下等部分对幽门 ( 图 1a , 点式行). </li>
	<li> 在结扎两个极点 ( 图 1b ) 后, 允许2分钟观察时间以确保没有出血. 
	注意: 在这个过程中, 避免结扎肾上腺、肾动脉和输尿管是非常重要的。如果左输尿管被结扎, 老鼠将在第二步手术中切除右肾后死亡。结扎力可以限制血流到两极, 而不会引起肾出血。在结扎后的 1-2 分钟内, 两极因血液流向两极的极限而变色. </li>
	<li> 将左肾推回到原来的位置。分别用4-0 和3-0 丝缝合线将肌肉和皮肤闭合。将抗生素软膏应用于手术区, 以更好的恢复和消毒. </li>
	<li> 结扎后, 将鼠标放在笼中, 将其放在一个带有热灯的笼子中, 使寝具区域接近37和 #176; C (用温度计监测), 然后将动物放在单独的笼子里. 
	注意: 监测鼠标, 直到它已经恢复足够的意识, 以维护胸骨卧床. </li>
	<li> 允许在恢复过程中方便地获得水和食物。管理青霉素和丁丙诺啡, 每12小时后头3天, 以防止感染和疼痛. 
	注意: 老鼠应该在 2-3 天内从手术创伤中恢复过来, 并且应该回到正常的饮食、饮酒和运动中。此时, 将鼠标逐个进行完全恢复。必须对以下情况进行监测: 饮酒、进食、行走方式、笨拙的步态、驼背、眼部和/或鼻腔分泌物以及攻击性行为。安乐如果它显示任何癫痫发作、昏迷、无法治愈的感染、行走困难、步态丧失、无法进食和饮水, 以及15% 的术后体重损失. </li>
</ol> 3。第二步手术: 右肾切除术 <ol> <li> 七天后, 在步骤2.1 到2.10 中将右肾暴露为左肾, 除非将鼠标放在其左侧. </li>
	<li> 将右肾放在亚麻布上, 用钝钳将周围的脂肪和结缔组织清除干净. </li>
	<li> 识别肾动脉和静脉, 然后将两个连字 (3-0 丝) 分别放置在血管周围的一个松散结。轻轻地挑逗结缔组织和肾上腺腺体从肾脏的上端向中间的幽门的肾脏使用钝钳。轻轻地将输尿管从下端移出, 并将所有的血管系在一起 (3-0 丝) 而不会造成出血. </li>
	<li> 将两个松散的连字节沿容器移动。一个朝向腹主动脉一侧, 另一个朝向肾侧. </li>
	<li> 首先将结扎结 (朝向腹主动脉侧) 与双节结; 实心结会改变右肾的颜色。将结扎结 (朝向肾脏一侧) 系成双结。在这两个结之间留出足够的空间, 以便在两节之间切断血管而不割断结扎结. </li>
	<li> 切开两节之间的肾血管, 取出右肾。检查肾血管是否可能出血. </li>
	<li> 用无菌纱布烘干右肾, 称其为重. </li>
	<li> 用4-0 丝缝合线关闭肌肉切口, 然后用3-0 丝缝合线闭合皮肤切口. </li>
	<li> 将鼠标分别放在一个干净的单独的笼子里, 在笼子上放一盏热灯 (步骤 2.15), 以方便地获得水和食物, 至少24小时才能恢复。管理青霉素和丁丙诺啡每12小时3天, 以防止感染和疼痛. 
	注意: 老鼠应该在 2-3 天内从手术创伤中恢复过来, 并且应该回到正常的饮食、饮酒和运动中. </li>
	<li> 手术后, 每天两次监测小鼠三天, 然后每天一次进行实验。遵循步骤2.16 中描述的后监视和处理程序. </li>
</ol> 4。假手术 <ol> <li> 为假手术, 执行相同的手术程序, 包括肾脏暴露, 组织解剖, 伤口闭合, 但没有左肾极结扎或右肾切除. </li> </ol>
 5. 实验性尿毒症心肌病的评估 <ol> <li> 在牺牲前一天, 执行经胸超声心动图和捕获图像 18 。
	注: 在这里, 经胸超声心动图进行了鼠标处理表和直肠温度计。超声心动图图像被捕获使用18-38 兆赫工作频率传感器连接到一个成像系统。根据我们描述的 18 , 计算出相对壁厚 (RWT)、心肌性能指数 (MPI) 和左心室质量指数 (LVMI). </li>
	<li> 在牺牲时, 测量体重。根据制造商和 #39 的指示, 用一台小贩的离心机测量红细胞压积 (小贩)。用肝素包膜管制备血浆样品, 离心10分钟, 在 1500 x g 在4和 #176; C 在台式冷藏离心机。根据制造商和 #39 的说明, 使用套件测量胱 C、肌酐和发髻。重复执行测量. </li>
	<li> 在实验结束时, 麻醉用氯胺酮 (90 毫克/千克, ip) 和 xylaxine (10 毫克/千克, ip) 和切口的牺牲。从胸部取出后测量心脏重量。准备心脏 (LV) 和肾脏匀, 以及 Type-1 胶原蛋白 (COL-1) 的测定, 磷酸化的 c Src 和蛋白质羰基, 如前所述, 18 , 19 . </li>
</ol>

<ol>
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作者没有什么可透露的。

大鼠 5/6th PNx 模型被广泛用于研究 CKD。由于小鼠的肾脏体积较小, 传统的动脉结扎和极切除术在小鼠模型中具有很大的挑战性, 可能造成高死亡率和意外出血/失血。
我们采用了小鼠 PNx 模型与极结扎, 以克服出血/失血。该 PNx 模型耗时少, 成活率高, 重现性好。这种极结扎模型在四周后时发展了人尿毒症心肌病的表型变化, 从而为研究其机制和治疗靶点提供了一个模型。...

CKD, 也被称为慢性肾衰竭, 是随着时间的推移肾脏功能的逐步丧失, 最终发展成永久性肾衰竭。CKD, 从早期肾脏疾病状态到终末期肾脏疾病 (末期), 是心血管疾病事件和死亡率的一个很大的危险因素, 并逐步发展到临床表型称为 "尿毒症心肌病"1, 2,3。CKD 或末期患者的尿毒症心肌病与心血管异常有关, 主要是由于左心室 (lv) 压力和/或容积超负荷导致的, 导致 lv 肥厚、lv 扩张和 lv 收缩功能障碍4 ,5,6。心脏纤维化是尿毒症心肌病的另一种常见的病理过程, 它能降低心脏顺应性, 导致左心室舒张功能障碍。严重的心脏纤维化可能导致心脏猝死甚至在那些没有心脏症状的患者中7。
5/6th PNx 是一种常用的 CKD 动物模型, 用于动物研究, 包括肾衰竭、尿毒症心肌病和高血压。PNx 是通过消融5/6 肾实质来实现的。大鼠模型最初是用两种最常用的手术切除或梗塞的方法开发的。大鼠 PNx 模型是一个非常有用的模型来研究尿毒症心肌实质升高血压, 心肌肥厚和舒张功能受损。后来, 老鼠 PNx 模型, 操作与相似的技术作为鼠模型, 被开发了由于广泛的可及性在小鼠系统做基因操纵。
有文献记载, 全身氧化剂应激是临床和实验性尿毒症心肌病的一个恒定特征8,9。此外, 氧化剂的压力有助于尿毒症综合征10, 并在尿毒症心肌病的发病机制中起关键作用111213。在这一点上, 我们已经证明了啮齿动物 5/6th PNx 模型导致尿毒症心肌病的生理、形态和生化特征1415,16, 17. 在这里描述的鼠标 PNx 模型中, PNx 操作的小鼠产生了明显的氧化应激, 至少部分由钠/钾 atp 酶信号传导, 这在 PNx 介导的尿毒症心肌病中是至关重要的。钠/钾 atp 酶信号的衰减不仅降低了氧化放大, 而且还改善了 PNx 介导的实验性尿毒症心肌病的型变化18。

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			<td>Hilltop Lab Animals Inc.</td>
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a mouse 5/6th nephrectomy model that induces experimental uremic cardiomyopathy

慢性肾脏疾病 (CKD) 是心血管疾病事件和死亡率的一个很大的危险因素, 并逐步发展到临床表型称为 "尿毒症心肌病"。我们在这里描述一个实验性 CKD 小鼠模型, 命名为 5/6th部分肾切除 (PNx) 与极结扎, 在四周后的发展为尿毒症心肌病。这种 PNx 模型是由 two-step 手术进行的。在第一步手术中, 左肾的两极都结扎了。在第一步手术7天后进行的两次手术中, 右肾切除。对于假手术, 同样的手术程序进行, 但没有极结扎左肾或切除右肾。与其他方法相比, 手术过程更容易、更耗时。然而, 残余功能性肾肿块不像肾动脉结扎那样容易控制。手术后四周, 与假手术小鼠相比, PNx 小鼠肾功能受损, 贫血, 心肌肥厚, 心脏纤维化, 心脏收缩和舒张功能下降。

数据表明, 此修改后的 5/6 th PNx 模型是一种简单有效的研究尿毒症心肌病的模型。在四周后, 该 PNx 模型显示肾功能受损, 贫血, 心肌肥厚, 心脏纤维化, 并降低心脏收缩和舒张功能。结果概述如下。
在四周的后, PNx 小鼠除了心脏形态学和生化变化, 与人类尿毒症心肌病的表型相一致外, 还发育受损的肾功能。?...

Watch this Scientific Journal Video about A Mouse 5/6th Nephrectomy Model That Induces Experimental Uremic Cardiomyopathy at JoVE.com

A Mouse 5/6th Nephrectomy Model That Induces Experimental Uremic Cardiomyopathy

这份手稿提供了一个详细的 two-step 手术程序执行小鼠 5/6th部分肾切除 (PNx) 与极结扎。手术后四周, 与假手术小鼠相比, PNx 小鼠肾功能受损, 贫血, 心肌肥厚, 心脏纤维化, 心脏收缩和舒张功能下降。

一种诱导实验性尿毒症心肌病的小鼠 5/6th肾切除模型

Chronic kidney disease (CKD) is a great risk factor for cardiovascular disease events and mortality, and progressively develops to the clinical phenotype ...

a-mouse-56th-nephrectomy-model-that-induces-experimental-uremic

Research

JoVE Journal

Biology

A Mouse 5/6th Nephrectomy Model That Induces Experimental Uremic Cardiomyopathy

20.2K 次观看.  Marshall University. 这份手稿提供了一个详细的 two-step 手术程序执行小鼠 5/6th部分肾切除 (PNx) 与极结扎。手术后四周, 与假手术小鼠相比, PNx 小鼠肾功能受损, 贫血, 心肌肥厚, 心脏纤维化, 心脏收缩和舒张功能下降。

视频: A Mouse 5/6th Nephrectomy Model That Induces Experimental Uremic Cardiomyopathy

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

活体成像和治疗卵巢癌的原位小鼠模型

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

科研

生物学

Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal

Pluripotency and self-renewal are two defining characteristics of embryonic stem cells (ES cells). Understanding the underlying molecular mechanism will greatly facilitate the use of ES cells for developmental biology studies, disease modeling, drug discovery, and regenerative medicine (reviewed in 1,2).
To expedite the identification and characterization of novel regulators of ES cell maintenance and self-renewal, we developed a fluorescence reporter-based assay to quantitatively measure the self-renewal status in mouse ES cells using the Oct4GiP cells 3. The Oct4GiP cells express the green fluorescent protein (GFP) under the control of the Oct4 gene promoter region 4,5. Oct4 is required for ES cell self-renewal, and is highly expressed in ES cells and quickly down-regulated during differentiation 6,7. As a result, GFP expression and fluorescence in the reporter cells correlates faithfully with the ES cell identity 5, and fluorescence-activated cell sorting (FACS) analysis can be used to closely monitor the self-renewal status of the cells at the single cell level 3,8.
Coupled with RNAi, the Oct4GiP reporter assay can be used to quickly identify and study regulators of ES cell maintenance and self-renewal 3,8. Compared to other methods for assaying self-renewal, it is more convenient, sensitive, quantitative, and of lower cost. It can be carried out in 96- or 384-well plates for large-scale studies such as high-throughput screens or genetic epistasis analysis. Finally, by using other lineage-specific reporter ES cell lines, the assay we describe here can also be modified to study fate specification during ES cell differentiation.

We describe a fluorescence reporter assay to quickly identify and characterize genes that regulate mouse embryonic stem cell maintenance and self-renewal.

Oct4GiP记者试验研究小鼠胚胎干细胞维持和自我更新的基因调节

Pluripotency and self-renewal are two defining characteristics of embryonic stem cells (ES cells). Understanding the underlying molecular mechanism will ...

An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for inclusion and exclusion of data points, and computer-aided pattern recognition to support the interpretation and findings of the analysis. It has become increasingly important to be able to measure fluorescence images constructed from three-dimensional (3D) datasets in order to be able to capture the complexity of cellular dynamics and understand the basis of cellular plasticity within biological systems. Sophisticated microscopy instruments have permitted the visualization of 3D fluorescence images through the acquisition of multispectral fluorescence images and powerful analytical software that reconstructs the images from confocal stacks that then provide a 3D representation of the collected 2D images. Advanced design-based stereology methods have progressed from the approximation and assumptions of the original model-based stereology1 even in complex tissue sections2. Despite these scientific advances in microscopy, a need remains for an automated analytic method that fully exploits the intrinsic 3D data to allow for the analysis and quantification of the complex changes in cell morphology, protein localization and receptor trafficking. 
Current techniques available to quantify fluorescence images include Meta-Morph (Molecular Devices, Sunnyvale, CA) and Image J (NIH) which provide manual analysis. Imaris (Andor Technology, Belfast, Northern Ireland) software provides the feature MeasurementPro, which allows the manual creation of measurement points that can be placed in a volume image or drawn on a series of 2D slices to create a 3D object. This method is useful for single-click point measurements to measure a line distance between two objects or to create a polygon that encloses a region of interest, but it is difficult to apply to complex cellular network structures. Filament Tracer (Andor) allows automatic detection of the 3D neuronal filament-like however, this module has been developed to measure defined structures such as neurons, which are comprised of dendrites, axons and spines (tree-like structure). This module has been ingeniously utilized to make morphological measurements to non-neuronal cells3, however, the output data provide information of an extended cellular network by using a software that depends on a defined cell shape rather than being an amorphous-shaped cellular model. To overcome the issue of analyzing amorphous-shaped cells and making the software more suitable to a biological application, Imaris developed Imaris Cell. This was a scientific project with the Eidgen&ouml;ssische Technische Hochschule, which has been developed to calculate the relationship between cells and organelles. While the software enables the detection of biological constraints, by forcing one nucleus per cell and using cell membranes to segment cells, it cannot be utilized to analyze fluorescence data that are not continuous because ideally it builds cell surface without void spaces. To our knowledge, at present no user-modifiable automated approach that provides morphometric information from 3D fluorescence images has been developed that achieves cellular spatial information of an undefined shape (Figure 1). 
We have developed an analytical platform using the Imaris core software module and Imaris XT interfaced to MATLAB (Mat Works, Inc.). These tools allow the 3D measurement of cells without a pre-defined shape and with inconsistent fluorescence network components. Furthermore, this method will allow researchers who have extended expertise in biological systems, but not familiarity to computer applications, to perform quantification of morphological changes in cell dynamics.

We developed a software platform that utilizes Imaris Neuroscience, ImarisXT and MATLAB to measure the changes in morphology of an undefined shape taken from three-dimensional confocal fluorescence of single cells. This novel approach can be used to quantify changes in cell shape following receptor activation and therefore represents a possible additional tool for drug discovery.

细胞形态变化，从三维荧光图像量化的分析工具，

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for ...

Strategies for Tracking Anastasis, A Cell Survival Phenomenon that Reverses Apoptosis

Anastasis (Greek for &#8220;rising to life&#8221;) refers to the recovery of dying cells. Before these cells recover, they have passed through important checkpoints of apoptosis, including mitochondrial fragmentation, release of mitochondrial cytochrome c into the cytosol, activation of caspases, chromatin condensation, DNA damage, nuclear fragmentation, plasma membrane blebbing, cell shrinkage, cell surface exposure of phosphatidylserine, and formation of apoptotic bodies. Anastasis can occur when apoptotic stimuli are removed prior to death, thereby allowing dying cells to reverse apoptosis and potentially other death mechanisms. Therefore, anastasis appears to involve physiological healing processes that could also sustain damaged cells inappropriately. The functions and mechanisms of anastasis are still unclear, hampered in part by the limited tools for detecting past events after the recovery of apparently healthy cells. Strategies to detect anastasis will enable studies of the physiological mechanisms, the hazards of undead cells in disease pathology, and potential therapeutics to modulate anastasis. Here, we describe effective strategies using live cell microscopy and a mammalian caspase biosensor&#160;for identifying and tracking anastasis in mammalian cells.

The term anastasis refers to the phenomenon in which dying cells reverse a cell suicide process at a late stage, repair themselves, and ultimately survive. Here we demonstrate protocols for detecting and tracking cells that undergo anastasis.

策略跟踪Anastasis，A细胞存活的现象，细胞凋亡反转

Anastasis (Greek for &#8220;rising to life&#8221;) refers to the recovery of dying cells. Before these cells recover, they have passed through important ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

胚胎干细胞的跨内细胞质量注射导致嵌合体率增高

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

A Mouse Model of Intestinal Partial Obstruction

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including tumorous growths. However, the cellular remodeling mechanisms involved in, and caused by, intestinal obstructions are poorly understood. Several animal models of intestinal obstructions have been developed, but the mouse model is the most cost/time effective. The mouse model uses the surgical implantation of an intestinal partial obstruction (PO) that has a high mortality rate if it is not performed correctly. In addition, mice receiving PO surgery fail to develop hypertrophy if an appropriate blockade is not used or not properly placed. Here, we describe a detailed protocol for PO surgery which produces reliable and reproducible intestinal obstructions with a very low mortality rate. This protocol utilizes a surgically placed silicone ring that surrounds the ileum which partially blocks digestive movement in the small intestine. The partial blockage makes the intestine become dilated due to the halt of digestive movement. The dilation of the intestine induces smooth muscle hypertrophy on the oral side of the ring that progressively develops over 2 weeks until it causes death. The surgical PO mouse model offers an in vivo model of hypertrophic intestinal tissue useful for studying pathological changes of intestinal cells including smooth muscle cells (SMC), interstitial cells of Cajal (ICC), PDGFR&#945;+, and neuronal cells during the development of intestinal obstruction.

Intestinal obstructions are a partial or complete blockage of the intestine that can cause severe abdominal pain, nausea, vomiting, and preventing the passage of stool. This procedure for creating intestinal partial obsructions in mice is reliable in studying the mechanisms underlying pathological cell growth and death in the intestine.

小肠部分梗阻的小鼠模型

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including ...

Purification of Platelets from Mouse Blood

Platelets are purified from whole blood to study their functional properties, which should be free from red blood cells (RBC), white blood cells (WBC), and plasma proteins. We describe here purification of platelets from mouse blood using three-fold more iohexol gradient medium relative to blood sample volume and centrifugation in a swinging bucket rotor at 400 x g for 20 min at 20 &#176;C. The recovery/yield of the purified platelets were 18.2-38.5%, and the purified platelets were in a resting state, which did not contain any significant number of RBC and WBC. The purified platelets treated with thrombin showed up to 93% activation, indicating their viability. We confirmed that the purified platelets are sufficiently pure using flow cytometric and microscopic evaluation. These platelets can be used for gene expression, activation, granule release, aggregation, and adhesion assays. This method can be used for purification of platelets from the blood of other species as well.

Here, mouse blood was collected in the presence of an anti-coagulant. The platelets were purified by iohexol gradient medium using low speed centrifugation. The platelets were activated with thrombin to investigate if they were viable. The quality of the purified platelets was analyzed by flow cytometry and microscopy.

小鼠血液中血小板的纯化

Platelets are purified from whole blood to study their functional properties, which should be free from red blood cells (RBC), white blood cells (WBC), ...

A Laser Capture Microdissection Protocol That Yields High Quality RNA from Fresh-frozen Mouse Bones

RNA yield and integrity are decisive for RNA analysis. However, it is often technically challenging to maintain RNA integrity throughout the entire laser capture microdissection (LCM) procedure. Since LCM studies work with low amounts of material, concerns about limited RNA yields are also important. Therefore, an LCM protocol was developed to obtain sufficient quantity of high-quality RNA for gene expression analysis in bone cells. The effect of staining protocol, thickness of cryosections, microdissected tissue quantity, RNA extraction kit, and LCM system used on RNA yield and integrity obtained from microdissected bone cells was evaluated. Eight-&#181;m-thick frozen bone sections were made using an adhesive film and stained using a rapid protocol for a commercial LCM stain. The sample was sandwiched between a polyethylene terephthalate (PET) membrane and the adhesive film. An LCM system that uses gravity for sample collection and a column-based RNA extraction method were used to obtain high quality RNAs of sufficient yield. The current study focusses on mouse femur sections. However, the LCM protocol reported here can be used to study in situ gene expression in cells of any hard tissue in both physiological conditions and disease processes.

A laser capture microdissection (LCM) protocol was developed to obtain sufficient quantity of high-quality RNA for gene expression analysis in bone cells. The current study focusses on mouse femur sections. However, the LCM protocol reported here can be used to study gene expression in cells of any hard tissue.

从新鲜冷冻的小鼠骨骼中产生高质量RNA的激光捕获微解剖协议

RNA yield and integrity are decisive for RNA analysis. However, it is often technically challenging to maintain RNA integrity throughout the entire laser ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

质谱分析，用于识别 EYFP 标记 CENP-A （EYFP-CENP-A） 的泛化

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

A Mouse Model of Lumbar Spine Instability

Intervertebral disc degeneration (IDD) is a common pathological change leading to low back pain. Appropriate animal models are desired for understanding the pathological processes and evaluating new drugs. Here, we introduced a surgically induced lumbar spine instability (LSI) mouse model that develops IDD starting from 1 week post operation. In detail, the mouse under anesthesia was operated by low back skin incision, L3&#8211;L5 spinous processes exposure, detachment of paraspinous muscles, resection of processes and ligaments, and skin closure. L4&#8211;L5 IVDs were chosen for the observation. The LSI model develops lumbar IDD by porosity and hypertrophy in endplates at an early stage, decrease in intervertebral disc volume, shrinkage in nucleus pulposus at an intermediate stage, and bone loss in lumbar vertebrae (L5) at a later stage. The LSI mouse model has the advantages of strong operability, no requirement of special equipment, reproducibility, inexpensive, and relatively short period of IDD development. However, LSI operation is still a trauma that causes inflammation within the first week post operation. Thus, this animal model is suitable for study of lumbar IDD.

We developed a lumbar intervertebral disc degeneration mouse model by resection of L3&#8211;L5 spinous processes along with supra- and inter-spinous ligaments and detachment of paraspinous muscles.

腰椎不稳定的鼠标模型

Intervertebral disc degeneration (IDD) is a common pathological change leading to low back pain. Appropriate animal models are desired for understanding ...

一种诱导实验性尿毒症心肌病的小鼠 5/6^th肾切除模型

摘要

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此视频中的章节

活体成像和治疗卵巢癌的原位小鼠模型

Oct4GiP记者试验研究小鼠胚胎干细胞维持和自我更新的基因调节

细胞形态变化，从三维荧光图像量化的分析工具，

策略跟踪Anastasis，A细胞存活的现象，细胞凋亡反转

胚胎干细胞的跨内细胞质量注射导致嵌合体率增高

小肠部分梗阻的小鼠模型

小鼠血液中血小板的纯化

从新鲜冷冻的小鼠骨骼中产生高质量RNA的激光捕获微解剖协议

质谱分析，用于识别 EYFP 标记 CENP-A （EYFP-CENP-A）的泛化

腰椎不稳定的鼠标模型

一种诱导实验性尿毒症心肌病的小鼠 5/6th肾切除模型

摘要

探索更多视频

此视频中的章节

活体成像和治疗卵巢癌的原位小鼠模型

Oct4GiP记者试验研究小鼠胚胎干细胞维持和自我更新的基因调节

细胞形态变化，从三维荧光图像量化的分析工具，

策略跟踪Anastasis，A细胞存活的现象，细胞凋亡反转

胚胎干细胞的跨内细胞质量注射导致嵌合体率增高

小肠部分梗阻的小鼠模型

小鼠血液中血小板的纯化

从新鲜冷冻的小鼠骨骼中产生高质量RNA的激光捕获微解剖协议

质谱分析，用于识别 EYFP 标记 CENP-A （EYFP-CENP-A） 的泛化

腰椎不稳定的鼠标模型

一种诱导实验性尿毒症心肌病的小鼠 5/6^th肾切除模型

质谱分析，用于识别 EYFP 标记 CENP-A （EYFP-CENP-A）的泛化