作者感谢 Linnea Tankred 和戴安娜 Rydholm 的畜牧业。

BALB/c 的两个性别的老鼠幼崽是按照在眼科和视力研究中使用动物的声明使用的, 这些议定书是由斯德哥尔摩伦理动物研究委员会批准的。老鼠被安置在凋落物中, 连同哺乳母亲, 每天有12小时/夜间循环, 免费获得食物和水, 每天都进行监测。
注: 在手术过程中, 小鼠在麻醉下使用挥发性异氟醚。眼部药膏在眼部手术中不鼓励, 因为它们可能会干扰治疗和使用的物质。如有必要, 为防止干眼, 可应用一滴无菌生理盐水。虽然葡萄膜黑色素穿刺是自我愈合, 在葡萄膜黑色素穿刺期间采取护理, 以确保不育的手术器械。术后治疗包括经皮下生理盐水溶出, 及盐酸丁卡因眼部局部用药镇痛。幼崽被允许恢复到胸骨卧床在一个暖气垫, 然后返回到护理母亲, 在一个干净的笼子。与同样的哺乳母亲保持了垃圾, 以避免压力。
1. 麻醉
<ol><li>准备麻醉感应室, 管理异氟醚麻醉, 并确保小鼠面罩也被耦合。 注: 执行所有动物实验与所有适用的道德许可协议。</li>
	<li>在大气空气中预先填充3-4% 挥发性异氟醚的感应腔。 注: 根据道德许可, 除异氟醚以外的其他麻醉物质可以使用。应在通风的工作台或通风罩上进行挥发性麻醉程序。</li>
	<li>在感应室放置一个产后 (P) 12.5 天的 BALB/c 小鼠幼犬。确认麻醉器具已准备好将异氟醚送到感应室。 注意: 老鼠幼崽是脆弱的, 最好处理的颈背。将幼崽放在一起作为垃圾或与哺乳母亲避免压力。</li>
	<li>让幼犬完全麻醉。</li>
	<li>轻轻地, 拿起小狗的颈背。 注: 保持感应室在任何时候关闭, 以安全和稳定的感应。</li>
	<li>把鼠标转移到老鼠面具上。移动鼠标时, 一定要将感应腔内的麻醉流转换为面罩。</li>
	<li>执行脚趾捏, 以确认麻醉。</li>
</ol>2. 外科立体镜穿刺程序
<ol><li>将鼠标定位在侧卧床位置。确认掩模定位良好, 使鼠标眼睛在立体镜的视野中。</li>
	<li>轻轻旋转鼠标头, 使眼睛朝向立体镜。 注意: 眼睛的清晰焦点应该在立体镜下实现。对于定位, 建议放大 16X, 而40X 应用于执行手术程序。如果道德许可允许, 修剪胡须的尖端可以促进眼睛的可视化。</li>
	<li>使用小栓钳, 小心地突出幼犬的眼睛, 施加向下的压力, 眼睑背部和腹部的眼睛。</li>
	<li>保持一个温柔, 但坚定的持有小狗的眼睛盖子, 用小型镊子。 注意: 推荐用非显性手进行眼突出和盖子, 因为主导手应执行穿刺程序。</li>
	<li>使用立体镜定位角膜缘。白化 BALB/c 的角膜缘可以很容易地识别由圆形血管丛后角膜。</li>
	<li>用0.25 毫米直径 (30 克) 斜面针, 执行小葡萄膜黑色素穿刺, 靠近 uvea 的后角膜缘限制。只使用针的尖端, 不超过一半的斜角 (相当于0.5 毫米), 刺穿 uvea。如果正确执行, 葡萄膜黑色素穿孔是自密封的, 但人工注射的研究物质可以通过相同的穿刺伤口管理。 注: 如有小鼠眼解剖学经验, 葡萄膜黑色素穿刺在睫状体与锯缘之间执行。在执行葡萄膜黑色素穿刺时必须小心, 以避免晶状体破裂。</li>
	<li>执行第二个葡萄膜黑色素穿刺在对面的眼睛从第一个穿刺地点。优化穿刺的距离和位置;考虑12和6点穿孔, 12 点的背部尽可能。</li>
	<li>每四天重复一次葡萄膜黑色素穿刺程序, 直到 P24.5。在每一个连续的葡萄膜黑色素穿刺前, 监视动物的状态特别注意在葡萄膜黑色素穿刺中由于晶状体损伤而出现的外伤性白内障, 或明显减少的眼压。目的是执行重复穿刺在相同的位置, 如前穿刺的最佳效果。</li>
</ol>3. 无创体内监测
<ol><li>在每个实验日的穿刺或注射前, 麻醉鼠标进行上述操作。</li>
	<li>用鼠标在侧卧床位置, 轻轻地凸出眼睛。</li>
	<li>以虹膜血管为中心, 以手术立体镜。</li>
	<li>拍一张虹膜脉片的照片, 相机安装在手术立体镜上。 注意: 虽然没有必要, 诱导减数分裂有助于规范虹膜区域的每一个动物。在选择减数分裂诱导剂时考虑道德许可。仔细的聚焦虹膜血管将有助于进一步定量分析。建议使用40X 级。</li>
</ol>4. 术后护理
<ol><li>从啮齿目动物面具中取出 BALB 幼犬, 然后将其轻轻地转移到一个用手术垫分层的加热垫上。</li>
	<li>应用一滴1% 丁卡因被刺穿的眼睛。 注意: 根据道德许可, 还可以使用其他止痛方法。</li>
	<li>用皮下注射200µL 的无菌生理盐水溶液对动物进行水合。</li>
	<li>在干净的老鼠笼子里把幼崽还给哺乳妈妈。</li>
	<li>监视恢复过程。幼崽应该能够 deambulate 和返回到垃圾。</li>
</ol>5. 眼球摘除术
<ol><li>宫颈脱位弄死动物。在立体镜下进行摘除和解剖, 以更好地可视化程序。 注: 可使用不同的安乐死程序。建议严格遵守道德许可。</li>
	<li>把眼睑拉开, 以改善眼睛的接触。</li>
	<li>用弯曲的波恩眼剪刀做一个小切口 (大约 0.5 cm) 在眼睛盖子的眼角附近。</li>
	<li>使用暴露的眼周空间, 在轨道内的地球后面插入微栓钳。</li>
	<li>抓住眼睛周围的组织, 轻轻地向上拉。避免抓住眼球, 使用靠近眼窝的周围组织。</li>
	<li>在眼眶内插入弯曲的波恩眼剪刀。</li>
	<li>切开周围的组织直到眼睛从插座中释放出来。 注意: 这种解剖技术保持眼部解剖和好处后分析。</li>
	<li>继续从眼睛中取出多余的组织。去除多余的组织可以在修复后进行, 如果首选。 注: BALB 小鼠黑色素缺乏, 因此, 为了增加对比度, 建议采用深色背景来促进解剖程序。</li>
	<li>在充满无菌磷酸盐缓冲生理盐水 (PBS) 的培养皿中简单冲洗全眼。</li>
	<li>将眼睛转移到含有1.5 毫升4% 缓冲福尔马林溶液的2毫升管上。</li>
	<li>在室温下固定眼睛6小时。 注意: 固定能赋予组织的刚性, 有利于虹膜的解剖。固定时间应测试和调整不同的应用。</li>
</ol>6. 立体镜下虹膜解剖程序
<ol><li>用1.5 毫升的塑料巴斯德吸管 (或类似的) 从管子中取出固定液, 用新鲜的 PBS 冲洗眼睛三次。</li>
	<li>将固定的眼睛放在平滑的干燥表面 (解剖支架) 上, 前面的腔朝向向上。</li>
	<li>用斜面30克针, 在角膜缘后执行一个入口点。</li>
	<li>用小栓钳握住前段, 将 Clayman Vannas 直剪刀的一尖端插入先前创建的开口。 注: 推荐用非显性手控制钳, 并采用具有主导性的剪刀。</li>
	<li>当用栓钳转动眼睛时, 在角膜缘周围切开以去除眼睛的后段。 注意: 执行部分切口将允许剪刀的内尖端继续在组织中, 并大大促进过程。</li>
	<li>将前段朝向向下, 露出晶状体。</li>
	<li>用小钳抓住它, 然后向上拉, 小心地取下镜片。 注: 斜面针可用于穿刺和拉镜头。</li>
	<li>将前段与角膜朝向下, 垂直于视野的切口定位。</li>
	<li>用小栓钳轻轻抓住睫状体后部的组织。</li>
	<li>用 Clayman-Vannas 直剪刀, 进行修剪刚刚前的睫状体, 以消除小梁网和隔离虹膜。确认小梁网被移除;虹膜应该在角膜内移动。</li>
	<li>小心转移前眼罩与小栓钳, 持有角膜, 到一个96井板包含200µL 的 PBS。</li>
	<li>继续保持在井中的角膜和解剖虹膜通过冲洗与 PBS 与一个吸管。 注意: 如果虹膜仍然附着在角膜上, 一些小梁网可能存在, 而30克针的斜角可以用来帮助取代虹膜。</li>
	<li>用小栓钳将角膜从96井中取出, 并将孤立的虹膜放在井中进行进一步分析。</li>
	<li>将游离漂浮的虹膜样品存放在摄氏4摄氏度。 注意: 尽管是固定的, 但不建议长期存储虹膜自由浮动样品。</li>
</ol>7. 可能的实验性读出
<ol><li>为了可视化解剖虹膜中的血管, 对血小板内皮细胞黏附分子 (PECAM)-1 或 isolectin B4 等标记物采用自由浮动全组免疫组织化学染色方法。或者, 使用全身灌注血管染色, 如荧光标记的高分子量 dextrans。</li>
	<li>使用体内无创图像执行在硅片平面安装和量化完整的虹膜血管与适当的成像软件。 注意: 使用密度测量法或与血管分析软件9可以量化血管内的体内和体外。</li>
	<li>处理整个眼球的核酸和蛋白质提取, 其次是 PCR 或免疫印迹法方法, 进行分子分析的穿刺眼。 注意: 组织的分子分析最好用非固定组织进行, 但非固定虹膜的解剖是非常具有挑战性的, 而整个眼睛的使用在以前的研究中呈现出统计学意义的结果8。</li>
</ol>

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作者没有什么可透露的。

在本协议中, 提出了一种新的葡萄膜黑色素穿刺诱导虹膜血管反应的方法。穿刺触发伤口愈合机制和促进血管反应在虹膜10,11。这与眼部病理一致, 如民主共和国和 NVG, 在虹膜虹膜的临床情况下, 眼睛后段视网膜血管生成反应加剧, 虹膜的血管化增加12 ,13。
角膜是缺血性的, 并且具有特殊的胶原结构, 从而产生透明度。与此一致, 眼睛前段的血管造影是虹膜血管的可视化, 可通过体内无创方法1314的虹膜血管直接可视化实现。以前动物模型的虹膜新生血管依赖于大眼睛的动物, 其中执行复杂的手术干预15,16,17, 或通过人工注射特定血管生成物质18,19. 在这种方法中, 使用颜料缺陷 BALB/c 小鼠可以直接观察虹膜血管的体内, 并允许无创的定量的虹膜新生血管的小鼠, 从而提供了一个新的工具来研究体内的血管生成。
此外, 提出的小鼠虹膜解剖方法, 可以分析穿刺诱导虹膜新生血管的特定标记。在这里, 虹膜血管 illustratively 染色的 PECAM-1 抗体和可视化的荧光显微镜。自由浮动, 全挂载染色协议很容易实现和相当例行。此外, 该模型可进行血管灌注方法。血管特异染色给予虹膜血管结构的评估, 从而通过基于用户或软件分析的方法对虹膜新生血管9进行量化.如前面所描述的8,20, 虹膜新生血管的插图在15天后穿刺诱导后被评估。然而, 对虹膜血管化的影响可以观察早在4天后穿刺诱导, 这表明, 评估穿刺诱导虹膜新生血管模型可以进行不同的时间点, 根据具体实验要求。
值得注意的是, 这项议定书提出了一些挑战, 特别是与老鼠眼相对于其他动物模型的大小有关。在执行葡萄膜黑色素穿刺程序时应格外小心, 避免晶状体接触或损伤眼睛。观察眼部状况是至关重要的, 动物显示与实验相关的白内障或下降的眼压应排除和适当的安乐死。老鼠虹膜非常脆弱, 有些俗气。正确的解剖和分离组织是该议定书的另一个关键步骤。正确的分离从小梁网是必要的位移虹膜从前室。此外, 由于脆弱性, 虹膜分离需要固定的组织, 这损害了下游分子分析。然而, 对未固定全眼的分子分析已成功应用于所提出的方法8。
总之, 所描述的协议提出了一种新的穿刺诱导虹膜新生血管的小鼠模型。该模型具有允许体内无创性直接可视化血管生成的优点。此外, 小啮齿类动物的使用使所提出的模型对虹膜虹膜的研究具有吸引力, 因为它避免了对大眼睛生物的使用, 并伴随着伦理上的限制。此外, 该程序可以结合注射亲或抗血管生成物质通过自密封穿刺, 提高其作为一个新的模式,体内血管新生。

虹膜, 连同睫状体和脉络膜, 包括 uvea, 这是最具血管的眼睛组织。虹膜血管是维持眼睛前腔内稳态的必要条件。由于动脉和静脉之间有大量的吻合口连接, 虹膜血管不仅为虹膜本身提供养分和氧气供应, 而且对眼睛的整个前段1。
新血管的形成, 或预先存在的血管生成, 是生理过程的基础, 如发展和伤口愈合2。血管内皮生长因子 (VEGF) 和纤溶酶原激活剂抑制剂 (排) 等多种典型因素对血管生成有很好的调节作用, 并有多个炎症因子, 这些因素的不平衡会导致病理血管生成3。
在眼中, 新生血管是引起视力威胁的疾病的原因, 例如增生性糖尿病视网膜病变 (NVG) 和新生血管性青光眼。在这些眼部疾病中, 病灶新生血管通常位于视网膜组织中, 但眼后和前眼室炎症和血管生成因子的不平衡与虹膜虹膜有关,临床术语虹膜病理血管生成 4.这些病理表明, 成人虹膜的能力, 以接受血管生成。小鼠出生后眼血管发育不成熟, 产后持续成熟。这种发展的特点被利用在老鼠模型的氧诱导视网膜病变, 一个模型, 密切模仿的临床条件的早产儿视网膜病变5。此外, 血管生成和炎症在创伤愈合机制中起关键作用6, 伤口愈合本身也与血管生成模型7有关。
在本研究中, 我们描述了穿刺诱导的虹膜新生血管模型。葡萄膜黑色素穿刺在边缘附近进行, 通过触发创面愈合系统诱导虹膜新生血管。由于角膜的透明性, 虹膜血管可以通过无创方法在体内进行简单的活体分析。穿孔的眼睛呈现增加的血管床在虹膜, 这是与增加的纤溶酶激活和炎症标记8。该模型具有很大的潜力, 作为一种新的工具来研究血管新生和筛选血管生成化合物, 并允许直接在体内可视化血管形成过程。

<table>
	<tbody>
		<tr>
			<td>Bonn eye scissors</td>
			<td>Bausch &#38; Lomb</td>
			<td>23060</td>
			<td></td>
		</tr>
		<tr>
			<td>Clayman-Vannas curved scissors</td>
			<td>Bausch &#38; Lomb</td>
			<td>E3383 C</td>
			<td></td>
		</tr>
		<tr>
			<td>Clayman-Vannas straight scissors</td>
			<td>Bausch &#38; Lomb</td>
			<td>E3383 S</td>
			<td></td>
		</tr>
		<tr>
			<td>Objective adapter for camera</td>
			<td>Handcrafted</td>
			<td>N/A</td>
			<td>Or any system that allows adapting a camera to the microscope</td>
		</tr>
		<tr>
			<td>Heating Pad 100-110 watts</td>
			<td>Non Applicable</td>
			<td>N/A</td>
			<td>Available in pet/veterinarian stores</td>
		</tr>
		<tr>
			<td>Hypodermic 30g beveled needle</td>
			<td>KDM GmBH germany</td>
			<td>911914</td>
			<td></td>
		</tr>
		<tr>
			<td>Iphone 4S</td>
			<td>Apple</td>
			<td>Non Applicable</td>
			<td>Or other high resolution image acquistion device</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Baxter</td>
			<td>KDG 9623</td>
			<td></td>
		</tr>
		<tr>
			<td>McPherson tying forceps</td>
			<td>Bausch &#38; Lomb</td>
			<td>E1815 S</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro tying forceps</td>
			<td>Bausch &#38; Lomb</td>
			<td>63140</td>
			<td></td>
		</tr>
		<tr>
			<td>Minims tetracaine hydrochloride</td>
			<td>Bausch &#38; Lomb</td>
			<td>N/A</td>
			<td>1 % (w/v) Eye Drops</td>
		</tr>
		<tr>
			<td>Neutral-buffered formalin</td>
			<td>Bioreagens</td>
			<td>0018-40</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal saline solution</td>
			<td>Fresenius Kabi</td>
			<td>210352</td>
			<td>0.9 % (w/v) NaCl in injectable water</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline</td>
			<td>ThermoFisher Scientific</td>
			<td>10010023</td>
			<td>Balanced and buffered PBS pH 7.4</td>
		</tr>
		<tr>
			<td>Petri dish 10 cm</td>
			<td>Starstedt</td>
			<td>83.3902</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish 3 cm</td>
			<td>Starstedt</td>
			<td>83.3900</td>
			<td></td>
		</tr>
		<tr>
			<td>Safe Seal Tube 2.0 mL</td>
			<td>Starstedt</td>
			<td>72.685.200</td>
			<td>Or any eppendorf style tubes</td>
		</tr>
		<tr>
			<td>TC plate 96-well</td>
			<td>Starstedt</td>
			<td>83.3924</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer pipette 3.5 mL</td>
			<td>Starstedt</td>
			<td>86.1171</td>
			<td>Or any other Pasteur pipette style</td>
		</tr>
		<tr>
			<td>Univentor 400 anesthesia unit</td>
			<td>Univentor Limited</td>
			<td>N/A</td>
			<td>Or equivalent flow regulator with induction chamber and mask for volatile anesthesia</td>
		</tr>
		<tr>
			<td>Wild M650 surgical microscope</td>
			<td>Wild Heerbrugg</td>
			<td>N/A</td>
			<td>Or other surgical or magnifying stereoscope</td>
		</tr>
	</tbody>
</table>

puncture-induced iris neovascularization as a mouse model of rubeosis iridis

我们描述了穿刺诱导的虹膜新生血管模型作为无创性血管生成评价的一般模型。该模型还与靶向新生血管性青光眼有关, 这是糖尿病视网膜病变的一种视力威胁并发症。该方法以 BALB 小鼠的一系列自密封葡萄膜黑色素穿刺诱导虹膜血管反应为基础, 利用小鼠眼血管的产后成熟。老鼠幼崽从产后12.5 葡萄膜黑色素穿刺, 当幼崽自然睁开眼睛, 直到产后24.5 天。由于角膜的透明度, 虹膜血管可以通过无创的体内方法很容易地通过时间进行分析。此外, BALB 小鼠的半透明虹膜可 flatmounted 为详细的 immunohistologic 分析和最小的非特异背景染色。在该模型中, 血管生成主要由炎症和纤溶酶激活系统驱动。穿刺诱导模型是诱发小鼠虹膜新生血管的第一种, 具有直接无创性在体内分析血管生成过程的优势。此外, 该模型可与血管生成调节物质结合, 突出了其在血管新生研究中的潜力, 其中以体内为视角。

白化 BALB/c 小鼠幼崽在 P12.5 受到葡萄膜黑色素穿刺, 重复每第四天 (实验日 0, 4, 8, 12), 直到 P24.5。在 P27.5, 老鼠被安乐死和虹膜仔细解剖 (实验日 15)。在每一个实验日的每一个穿刺系列中, 用一台附有手术立体镜的照相机拍摄了老鼠眼的照片, 以评估虹膜血管反应的无创性评价。葡萄膜黑色素穿刺通过触发伤口愈合系统(图 1) 引起虹膜的血管反应。由于角膜和黑色素缺乏的 BALB 小鼠的透明度, 增加的虹膜血管从实验日 4 (P16.5) 就可以看到, 并在整个议定书期间强化。PECAM-1 的免疫组化图像表示, 与控制 (图 2) 相比, 穿刺眼睛虹膜中的总血管增多。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57398/57398fig1.jpg"> 图 1: 穿刺诱发的虹膜血管生成的无创监测.0, 4, 8, 12 和15的控制和刺穿眼睛的代表性图像。虹膜血管反应是明显的从4天开始在刺穿眼睛。刻度条 = 1 毫米.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57398/57398fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57398/57398fig2.jpg"> 图 2: 穿刺诱导眼中的虹膜血管反应.PECAM-1 染色的说明图像从15天虹膜, 显示为全视图和放大面积 (正方形), 从控制和刺穿眼睛。刻度条 = 400 µm (顶部);200µm (底部)。注意穿刺虹膜中血管分支的增加。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57398/57398fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Puncture-Induced Iris Neovascularization as a Mouse Model of Rubeosis Iridis at JoVE.com

Puncture-Induced Iris Neovascularization as a Mouse Model of Rubeosis Iridis

虹膜新生血管是缺血性视网膜疾病的常见并发症, 可能导致视力威胁的新生血管性青光眼。在这里, 我们描述了诱导实验性虹膜新生血管的小鼠协议, 可用于无创性评价血管生成调节物质。

穿刺诱导的虹膜新生血管虹膜虹膜小鼠模型的建立

We describe a model of puncture-induced iris neovascularization as a general model for noninvasive evaluation of angiogenesis. The model is also relevant ...

puncture-induced-iris-neovascularization-as-mouse-model-rubeosis

Nanyang Technological University

Research

JoVE Journal

Medicine

8.7K 次观看.  Karolinska Institutet. 虹膜新生血管是缺血性视网膜疾病的常见并发症, 可能导致视力威胁的新生血管性青光眼。在这里, 我们描述了诱导实验性虹膜新生血管的小鼠协议, 可用于无创性评价血管生成调节物质。

视频: Puncture-Induced Iris Neovascularization as a Mouse Model of Rubeosis Iridis

A Mouse Model of in Utero Transplantation

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus. For example, in utero transplantation (IUT) of hematopoietic stem cells has been used to successfully treat patients with severe combined immunodeficiency.1,2 In several other conditions, however, IUT has been attempted without success.3 Given these mixed results, the availability of an efficient non-human model to study the biological sequelae of stem cell transplantation and gene therapy is critical to advance this field. We and others have used the mouse model of IUT to study factors affecting successful engraftment of in utero transplanted hematopoietic stem cells in both wild-type mice4-7 and those with genetic diseases.8,9 The fetal environment also offers considerable advantages for the success of in utero gene therapy. For example, the delivery of adenoviral10, adeno-associated viral10, retroviral11, and lentiviral vectors12,13 into the fetus has resulted in the transduction of multiple organs distant from the site of injection with long-term gene expression. in utero gene therapy may therefore be considered as a possible treatment strategy for single gene disorders such as muscular dystrophy or cystic fibrosis. Another potential advantage of IUT is the ability to induce immune tolerance to a specific antigen. As seen in mice with hemophilia, the introduction of Factor IX early in development results in tolerance to this protein.14 
In addition to its use in investigating potential human therapies, the mouse model of IUT can be a powerful tool to study basic questions in developmental and stem cell biology. For example, one can deliver various small molecules to induce or inhibit specific gene expression at defined gestational stages and manipulate developmental pathways. The impact of these alterations can be assessed at various timepoints after the initial transplantation. Furthermore, one can transplant pluripotent or lineage specific progenitor cells into the fetal environment to study stem cell differentiation in a non-irradiated and unperturbed host environment.
The mouse model of IUT has already provided numerous insights within the fields of immunology, and developmental and stem cell biology. In this video-based protocol, we describe a step-by-step approach to performing IUT in mouse fetuses and outline the critical steps and potential pitfalls of this technique.

A Mouse Model of in Utero Transplantation

The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique

一个小鼠模型在子宫内移植

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

科研

医学

Mouse Complete Stasis Model of Inferior Vena Cava Thrombosis

Venous thromboembolism (VTE) includes both deep vein thrombosis (DVT) and pulmonary embolism (PE). In the United States (U.S.), the high morbidity and mortality rates make VTE a serious health concern 1-2. After heart disease and stroke, VTE is the third most common vascular disease 3. In the U.S. alone, there is an estimated 900,000 people affected each year, with 300,000 deaths occurring annually 3. A reliable in vivo animal model to study the mechanisms of this disease is necessary.
The advantages of using the mouse complete stasis model of inferior vena cava thrombosis are several. The mouse model allows for the administration of very small volumes of limited availability test agents, reducing costs dramatically. Most promising is the potential for mice with gene knockouts that allow specific inflammatory and coagulation factor functions to be delineated. Current molecular assays allow for the quantitation of vein wall, thrombus, whole blood, and plasma for assays. However, a major concern involving this model is the operative size constraints and the friability of the vessels. Also, due to the small IVC sample weight (mean 0.005 grams) it is necessary to increase animal numbers for accurate statistical analysis for tissue, thrombus, and blood assays such as real-time polymerase chain reaction (RT-PCR), western blot, enzyme-linked immunosorbent (ELISA), zymography, vein wall and thrombus cellular analysis, and whole blood and plasma assays 4-8.
The major disadvantage with the stasis model is that the lack of blood flow inhibits the maximal effect of administered systemic therapeutic agents on the thrombus and vein wall.

The mouse complete stasis model of inferior vena cava thrombosis yields quantifiable amounts of vein wall tissue and thrombus. It has proven useful for evaluating interactions between the vein wall and the occlusive thrombus and in assessing the progression from acute to chronic inflammation.

鼠标完成血瘀模型的下腔静脉血栓

Venous thromboembolism (VTE) includes both deep vein thrombosis (DVT) and pulmonary embolism (PE).  In the United States (U.S.), the high morbidity and ...

Rapid Point-of-Care Assay of Enoxaparin Anticoagulant Efficacy in Whole Blood

There is the need for a clinical assay to determine the extent to which a patient's blood is effectively anticoagulated by the low-molecular-weight-heparin (LMWH), enoxaparin. There are also urgent clinical situations where it would be important if this could be determined rapidly. The present assay is designed to accomplish this. We only assayed human blood samples that were spiked with known concentrations of enoxaparin. The essential feature of the present assay is the quantification of the efficacy of enoxaparin in a patient's blood sample by degrading it to complete inactivity with heparinase. Two blood samples were drawn into Vacutainer tubes (Becton-Dickenson; Franklin Lakes, NJ) that were spiked with enoxaparin; one sample was digested with heparinase for 5 min at 37 &deg;C, the other sample represented the patient's baseline anticoagulated status. The percent shortening of clotting time in the heparinase-treated sample, as compared to the baseline state, yielded the anticoagulant contribution of enoxaparin. We used the portable, battery operated Hemochron 801 apparatus for measurements of clotting times (International Technidyne Corp., Edison, NJ). The apparatus has 2 thermostatically controlled (37 &deg;C) assay tube wells. We conducted the assays in two types of assay cartridges that are available from the manufacturer of the instrument. One cartridge was modified to increase its sensitivity. We removed the kaolin from the FTK-ACT cartridge by extensive rinsing with distilled water, leaving only the glass surface of the tube, and perhaps the detection magnet, as activators. We called this our minimally activated assay (MAA). The use of a minimally activated assay has been studied by us and others. 2-4 The second cartridge that was studied was an activated partial thromboplastin time (aPTT) assay (A104). This was used as supplied from the manufacturer. The thermostated wells of the instrument were used for both the heparinase digestion and coagulation assays. The assay can be completed within 10 min. The MAA assay showed robust changes in clotting time after heparinase digestion of enoxaparin over a typical clinical concentration range. At 0.2 anti-Xa I.U. of enoxaparin per ml of blood sample, heparinase digestion caused an average decrease of 9.8% (20.4 sec) in clotting time; at 1.0 I.U. per ml of enoxaparin there was a 41.4% decrease (148.8 sec). This report only presents the experimental application of the assay; its value in a clinical setting must still be established.

Demonstration of a rapid quantitative assay of the inhibition of blood coagulation by the low-molecular-weight-heparin, enoxaparin. The contribution of enoxaparin is assessed by removing its influence through digestion with heparinase. A fuller description of the assay is detailed in our published paper.1 The assay still requires clinical confirmation.

快速点护理依诺肝素的抗凝血剂功效的测定全血中

There is the need for a clinical assay to determine the extent to which a patient's blood is effectively anticoagulated by the ...

Intraductal Injection of LPS as a Mouse Model of Mastitis: Signaling Visualized via an NF-&kappa;B Reporter Transgenic

Animal models of human disease are necessary in order to rigorously study stages of disease progression and associated mechanisms, and ultimately, as pre-clinical models to test interventions. In these methods, we describe a technique in which lipopolysaccharide (LPS) is injected into the lactating mouse mammary gland via the nipple, effectively modeling mastitis, or inflammation, of the gland. This simulated infection results in increased nuclear factor kappa B (NF-&kappa;B) signaling, as visualized through bioluminescent imaging of an NF-&kappa;B luciferase reporter mouse1.
Our ultimate goal in developing these methods was to study the inflammation associated with mastitis in the lactating gland, which often includes redness, swelling, and immune cell infiltration2,3. Therefore, we were keenly aware that incision or any type of wounding of the skin, the nipple, or the gland in order to introduce the LPS could not be utilized in our methods since the approach would likely confound the read-out of inflammation. We also desired a straight-forward method that did not require specially made hand-drawn pipettes or the use of micromanipulators to hold these specialized tools in place. Thus, we determined to use a commercially available insulin syringe and to inject the agent into the mammary duct of an intact nipple. This method was successful and allowed us to study the inflammation associated with LPS injection without any additional effects overlaid by the process of injection. In addition, this method also utilized an NF-&kappa;B luciferase reporter transgenic mouse and bioluminescent imaging technology to visually and quantitatively show increased NF-&kappa;B signaling within the LPS-injected gland4.
These methods are of interest to researchers of many disciplines who wish to model disease within the lactating mammary gland, as ultimately, the technique described here could be utilized for injection of a number of substances, and is not limited to only LPS.

Described here is a technique in which lipopolysaccharide is injected into the lactating mouse mammary gland via the nipple to simulate mastitis, a condition commonly caused by bacterial infection. Lipopolysaccharide injection results in increased nuclear factor kappa B (NF-&kappa;B) signaling, visualized through bioluminescent imaging of an NF-&kappa;B luciferase reporter mouse.

导管内可视化通过NF-κB记者的转基因小鼠模型的乳腺炎:信号注射LPS

Animal models of human disease are necessary in order to rigorously study stages of disease progression and associated mechanisms, and ultimately, as ...

The In ovo CAM-assay as a Xenograft Model for Sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for personalized medicine after stratification, explaining the current interest in developing a reproducible and low-cost xenotransplant model for this disease. The chick chorioallantoic membrane is a natural immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition, it is easily accessed, manipulated and imaged using optical and fluorescence stereomicroscopy. Histology further allows detailed analysis of heterotypic cellular interactions.
This protocol describes in detail the in ovo grafting of the chorioallantoic membrane with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines (Saos-2 and SW1353). The chick survival rates are up to 75%. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior. For localized grafting of single cell suspensions, ECM gel provides significant advantages over inert containment materials. The Ki67 proliferation index is related to the distance of the cells from the surface of the CAM and the duration of application on the CAM, the latter determining a time frame for the addition of therapeutic products.

The In ovo CAM-assay as a Xenograft Model for Sarcoma

The in ovo chorioallantoic membrane (CAM) is grafted with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior.

该<em&gt;在OVO</em&gt; CAM作为一个肉瘤Xenograftmodel的检测

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for ...

An Alkali-burn Injury Model of Corneal Neovascularization in the Mouse

Under normal conditions, the cornea is avascular, and this transparency is essential for maintaining good visual acuity. Neovascularization (NV) of the cornea, which can be caused by trauma, keratoplasty or infectious disease, breaks down the so called &lsquo;angiogenic privilege' of the cornea and forms the basis of multiple visual pathologies that may even lead to blindness. Although there are several treatment options available, the fundamental medical need presented by corneal neovascular pathologies remains unmet. In order to develop safe, effective, and targeted therapies, a reliable model of corneal NV and pharmacological intervention is required. Here, we describe an alkali-burn injury corneal neovascularization model in the mouse. This protocol provides a method for the application of a controlled alkali-burn injury to the cornea, administration of a pharmacological compound of interest, and visualization of the result. This method could prove instrumental for studying the mechanisms and opportunities for intervention in corneal NV and other neovascular disorders.

Neovascularization (NV) of the cornea can complicate multiple visual pathologies. Utilizing a controlled, alkali-burn injury model, a quantifiable level of corneal NV can be produced for mechanistic study of corneal NV and evaluation of potential therapies for neovascular disorders.

角膜新生血管在小鼠碱烧伤模型

Under normal conditions, the cornea is avascular, and this transparency is essential for maintaining good visual acuity. Neovascularization (NV) of the ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

脑Microslices的兴奋性刺激作为<em&gt;在体外</em&gt;中风模型

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

A Mouse Model for Laser-induced Choroidal Neovascularization

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) research. By administering targeted laser injury to the RPE and Bruch&#8217;s membrane, the procedure induces angiogenesis, modeling the hallmark pathology observed in neovascular AMD. 
First developed in non-human primates, the laser-induced CNV model has come to be implemented into many other species, the most recent of which being the mouse. Mouse experiments are advantageously more cost-effective, experiments can be executed on a much faster timeline, and they allow the use of various transgenic models. The miniature size of the mouse eye, however, poses a particular challenge when performing the procedure. Manipulation of the eye to visualize the retina requires practice of fine dexterity skills as well as simultaneous hand-eye-foot coordination to operate the laser. However, once mastered, the model can be applied to study many aspects of neovascular AMD such as molecular mechanisms, the effect of genetic manipulations, and drug treatment effects. 
The laser-induced CNV model, though useful, is not a perfect model of the disease. The wild-type mouse eye is otherwise healthy, and the chorio-retinal environment does not mimic the pathologic changes in human AMD. Furthermore, injury-induced angiogenesis does not reflect the same pathways as angiogenesis occurring in an age-related and chronic disease state as in AMD.
Despite its shortcomings, the laser-induced CNV model is one of the best methods currently available to study the debilitating pathology of neovascular AMD. Its implementation has led to a deeper understanding of the pathogenesis of AMD, as well as contributing to the development of many of the AMD therapies currently available.

Here, we present the mouse laser-induced choroidal neovascularization (CNV) protocol, an experimental model that re-creates the vascular hallmarks of neovascular age-related macular degeneration (AMD). Once mastered, it can reliably and effectively induce CNV as a model system to test various experimental measures.

小鼠模型的激光诱导脉络膜新生血管

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) ...

Tachycardia-Induced Cardiomyopathy As a Chronic Heart Failure Model in Swine

A stable and reliable model of chronic heart failure is required for many experiments to understand hemodynamics or to test effects of new treatment methods. Here, we present such a model by tachycardia-induced cardiomyopathy, which can be produced by rapid cardiac pacing in swine.
A single pacing lead is introduced transvenously into fully anaesthetized healthy swine, to the apex of the right ventricle, and fixated. Its other end is then tunneled dorsally to the paravertebral region. There, it is connected to an in-house modified heart pacemaker unit that is then implanted in a subcutaneous pocket. 
After 4 - 8 weeks of rapid ventricular pacing at rates of 200 - 240 beats/min, physical examination revealed signs of severe heart failure - tachypnea, spontaneous sinus tachycardia, and fatigue. Echocardiography and X-ray showed dilation of all heart chambers, effusions, and severe systolic dysfunction. These findings correspond well to decompensated dilated cardiomyopathy and are also preserved after the cessation of pacing.
This model of tachycardia-induced cardiomyopathy can be used for studying the pathophysiology of progressive chronic heart failure, especially hemodynamic changes caused by new treatment modalities like mechanical circulatory supports. This methodology is easy to perform and the results are robust and reproducible.

Here, we present a protocol to produce tachycardia-induced cardiomyopathy in swine. This model represents a potent way to study the hemodynamics of progressive chronic heart failure and the effects of applied treatment.

心动过速诱发的心肌病作为猪慢性心力衰竭模型的研究

A stable and reliable model of chronic heart failure is required for many experiments to understand hemodynamics or to test effects of new treatment ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...