The authors would like to thank Janelle Ryals, Rachel Supple, and Frank Wang for technical assistance. This work was supported by NIH grants R03 DK080182 (JAC), R01 DK099611 (JAC), R01 DK103872 (JAC), Center of Biomedical Research Excellence (COBRE) grant P20 GM104936 (JAC), start-up funds and core support from the Kansas Institutional Development Award (IDeA) P20 GM103418, core support from the Kansas IDDRC P30 HD002528, and The Madison and Lila Self Fellowship Program (ANP).

在这个协议中所描述的所有实验符合美国国立卫生研究院指南根据由堪萨斯大学医学中心机构动物护理和使用委员会的大学指定的准则。 1.新生儿母婴分离（NMS） <ol><li>日常监测大坝怀孕产仔胎。 注：出生的垃圾被认为是P0的一天。 </li><li>在P1，从家里取出笼坝并放入一个干净的二手集装箱。 </li><li>擦干净，戴手套的手之间的少数床上用品，以保持家笼的气味。 </li><li>加的深度为1 - 2厘米家笼寝具到干净，适当标记的2升玻璃烧杯中。添加约一半的任何附加富集寝具材料，是存在于家笼，例如 nestlet，皱纸，向玻璃烧杯中。 </li><li>轻轻放置所有幼仔的从单个窝，单独地在同一烧杯中。 </li><li>立即将在孵化器将烧杯在33℃，湿度50％保持180分钟。 </li><li>从二级容器中取出大坝，并返回到她家笼。回到家笼其适当的住房的位置在动物饲养中。 </li><li>重复此步骤，对每个垃圾进行网管，使用干净的手套和二级容器每窝/大坝。 </li><li>在180分钟间隔周期的结束，因为他们被拆除返回以相同的顺序将窝对他们的家笼中。 </li><li>检索是当天早些时候分离第一胎的家笼。从笼子中取出大坝并放入一个干净的二手集装箱。 </li><li>从孵化器中删除第一烧杯中，擦了一把干净的戴手套的手之间的床上用品，以保持家笼的气味，同时处理的幼崽。 </li><li>轻轻移动幼仔，分别从烧杯中的家笼。 </li><li>返回任何铀浓缩和床上用品留在烧杯中家笼。 </li><li>返回大坝从二级容器的家笼，并在动物饲养中返回到其适当的住房的位置。 </li><li>冲洗烧杯，用水，并将其返回到培养箱中。使用相同的烧杯整个分离步骤的每个垃圾。 </li><li>重复此步骤，对每个垃圾经历相同的顺序网管系统，因为他们被放置在孵化器。 </li><li>重复整个过程每窝经受网管每天通过P21。 </li><li>通过将同性别的同窝在一起，干净笼子完整的食物和水对断奶P22 NMS和天真的幼崽。幼稚幼仔应出生和收容在相同的条件网管小鼠下，但经历没有额外的处理的正常畜​​牧业任务之外。 </li></ol> 2. Perigenital机械感<ol><li>冯弗雷设备安装<ol><li>把老鼠的试验室，让他们适应环境30分钟，而留在家里的笼子。 </li><li>通过将吸收性焊升高丝网顶表（79厘米×28厘米），它提供了足够的空间，以允许研究者以从下侧接近而不惊人的小鼠，约55厘米的高度之下准备的测试区域。 </li><li>将多达12只，分别下清晰，穿孔塑料室（11厘米×5厘米×3.5厘米）的丝网桌子上。放置在腔室顶部上的重物，以防止小鼠逸出。 </li><li>适应的小鼠前逃避阈值评估到屏幕上的另外30分钟。 </li></ol></li><li> Perigenital机械感度评估<ol><li>执行使用了一组标准分级的von Frey单丝43的上下方法。使用以下单丝：1.65克，2.36克2.83克，3.22克，3.61克，4.08克，4.31克和4.74克。 <ol><li>握住3.22克单丝的基极与暴露在缩回单灯丝。 注：某些型号可能没有缩回单丝。 </li><li>位于所述探针，使得单丝被垂直定向并且当鼠标警报，不动的，而它的体重均匀地分布后爪之间将其应用到左或右阴囊侧，避免了中线，直至有轻微的弯曲在灯丝观察。 </li><li>保持在适当位置的单丝为10秒或直至动物显示了积极的响应。 注意：阳性反应被认为是一个轻快加加速度或跳跃响应于单丝应用或舔或朝向单丝咬行为。如果10秒单丝的应用程序在移动鼠标时不显示这些行为，单丝必须遵循最小1分钟长的休息时间重新测试。如果小鼠既不移动也不是10秒单丝应用期间出现任何上面列出的行为，它被认为是一个否定的响应。 </li><html><li>记录的反应，无论是正面或负面的，在实验室笔记本电脑或笔记本电脑，并在所有使用3.22克单丝剩余小鼠重复这一过程。 </li><li>使用下一个合适的单丝再次测试所有的老鼠。注意：如果鼠标展示在该3.22克单丝的否定响应，应用3.61克单丝以相同的方式，记录的响应。如果鼠标展示在3.22克单丝的积极响应，应用2.83克单丝和记录的响应。 </li></ol></li><li>继续测试使用下一个更大或更小的单丝各小鼠，在适当时进行额外的4个应用第一肯定响应观察后。 </li><li>回到老鼠的笼子。 </li><li>使用日志单位最终冯弗雷单丝中试系列应用价值中，Chaplan 等43所描述来计算50％G撤出门槛。 </li></ol></li></ol>标题“&gt; 3。酸化甲苯胺蓝肥大细胞染色<ol><li>组织处理<ol><li>从已经心内灌注冰-冷的4％低聚甲醛45小鼠解剖前列腺组织44。后缀的组织在4％多聚甲醛进行1小时，在RT，然后cryoprotect在30％的蔗糖在1×PBS中，在4℃CO / N。 </li><li>制备庚烷的小（30毫升）浴，并将其放置在干冰上。 </li><li>在冲洗1X PBS前列腺组织和干燥使用轻型擦拭。 </li><li>将前列腺组织进入冷的庚烷，直到它被冻结。 </li><li>立即将冷冻的前列腺组织中含有干冰上安装媒体和地方cryomold直到冻结。 </li><li>商店cryomolds在-20℃。 </li><li>横向切割前列腺组织成7使用低温恒温器微米厚的冰冻切片。 </li><li>放置8 - 12横跨组织的长度，到带正电的显微镜载玻片非串行冰冻切片。 </li><li>商店完成载玻片在-20℃直到进一步处理。 </li></ol></li><li>准备酸化甲苯胺蓝<ol><li>加入1克甲苯胺蓝O操作百毫升70％的乙醇和旋涡，直到在溶液中以制备1％的储液。原液可以储存在室温下长达三个星期。 </li><li>加入1克NaCl至100毫升的水在烧杯放在搅拌盘的顶部。 </li><li>调整用12 M盐酸直至0.5的pH范围的NaCl溶液的pH - 1.0实现。 </li><li>通过组合8毫升的甲苯胺蓝原液和32毫升1％的NaCl溶液中制备0.25％甲苯胺蓝工作溶液。吸液管上下以确保甲苯胺蓝原液的总掺入和混合使用涡流。该解决方案的工作必须做出新的，用后丢弃。 </li></ol></li><li>染色前列腺冷冻切片<ol><li>除去被从冷冻处理的幻灯片，并允许平衡至室温约30μm研究。 </li><li>通过单独浸渍幻灯片约1秒到50毫升锥形管中含有的1×PBS的足够体积，以确保在载玻片上的组织切片将被完全淹没洗组织切片。允许幻灯片空气干燥的纸巾。 </li><li>在含有足够0.25％的工作甲苯胺蓝溶液的玻璃染色缸地方滑动，以确保在载玻片上的组织切片将被完全淹没并孵育10 - 15分钟，同时在一个平台上摇杆设定为每分钟15转。 </li><li>浸幻灯片1X PBS约1秒至洗出多余的甲苯胺蓝。 </li><li>放置在一个滑动箱或机架滑轨以这样的方式，该滑动是在其两侧以允许排水。 </li><li>干的炉中的幻灯片，在37℃下进行至少2小时或O / N在室温。 </li><li>脱水幻灯片迅速使用酒精，使载片醇曝光之间完全干燥。干片由排在纸巾上一个第二擦背部和边缘用轻型擦拭。 <ol><li>浸泡在95％乙醇的幻灯片约1秒，并允许完全干燥。重复此步骤1 - 10次，直到组织比干燥更紫蓝色。 </li><li>沉浸在100％EtOH中的幻灯片大约1秒，并允许以完全干燥。重复此步骤1 - 10次，直到组织是黑暗中蓝色，而不是紫色。 </li></ol></li><li>通过将在100％二甲苯幻灯片3分钟固定的污渍。晾干。 </li><li>盖玻片的载玻片与甘油，PBS或二甲苯基永磁安装介质。沥干多余的介质，并储存在室温。 </li></ol></li><li>量化肥大细胞<ol><li>可视化的甲苯胺蓝染色的组织切片，在20X放大倍率下使用光学显微镜（ 图2）。 </li><li>算的可视化区域之内的本肥大细胞的总数和非脱颗粒和脱颗粒的肥大细胞区分。 40X尊离子可能是必要的，以确认在​​一些肥大细胞造粒状态。 注：非脱颗粒的肥大细胞表现出与没有或微弱的核轮廓和/或细胞周围没有颗粒挤压密实异染性。脱颗粒的肥大细胞显示出不太激烈异染，并有内细胞质和细胞边界以外的核和/或游离的颗粒的一个明显的清晰轮廓。 </li><li>继续评估当前组织切片的整体的非脱颗粒和脱颗粒的肥大细胞的总数。重复上至少有7个额外的单独章节此过程中，跨越长度的每个组织。 </li><li>根据以下等式计算脱颗粒（DG）的总的肥大细胞的百分比为每个组织/小鼠：（DG肥大细胞/总肥大细胞）×100。 </li></ol></li></ol>

The authors have nothing to disclose.

这个协议提供了方法用于使用新生儿应力，在网管系统的形式，以诱导症候表示的CP / CPPS的成年雄性小鼠。作为成年人，网管小鼠显示显著perigenital机械异常性疼痛，以及对肥大细胞脱粒的前列腺组织中的证据。使用NMS的是一种新颖的方法来开发的CP / CPPS的，因为它复制早期生活压力即常报道患有慢性盆腔疼痛12-14，临床前模型以及掺入一种非侵入性的诱导，如相对于更常用的，chemically-?...

慢性盆腔疼痛本身不是一种疾病，而是与经历的患者诊断为肠易激综合征（IBS）正在进行的自发和/或诱发疼痛，间质性膀胱炎/膀胱疼痛综合征（IC / PBS），慢性阴部疼痛相关的术语，或慢性前列腺炎/慢性骨盆疼痛综合征（CP / CPPS）。这些综合征常常伴发与共享许多特征在于它们具有无相关的病理或鉴定潜在的病因，虽然免疫系统，中枢神经系统和外周神经系统内的功能障碍已被证明对这些病症1的维护和发展作出贡献-3。慢性骨盆疼痛更可能呈现与症状的其他非盆腔相关功能性疼痛病症和心境障碍，包括焦虑，抑郁，和惊恐障碍4-6，这已经与下丘脑的改变的功能相关的垂体 -肾上腺（HPA）轴7-10。暴露于早期生活应激或创伤是用于开发HPA异常和相关慢性疼痛综合症10,11和，作为这种显著危险因素，患者的官能骨盆疼痛病症一个显著子集报告已经经历了不利的童年事件如虐待或忽视12-14。 新生儿母婴分离（NMS），其中涉及删除从他们的时间在断奶前一段在设定时间内大坝幼崽的啮齿动物模型已被用于在过去的二十年里，研究早期生活压力的结果。在一般情况下，网络管理系统已被证明能增加HPA轴活化，并产生焦虑样行为，通过直接影响基因表达的下丘脑内，以及扰乱从边缘结构15-18的下游调节。正确的HPA轴功能的干扰已被证明对提高大肠癌19-22贡献</sup&gt;和阴道显示的啮齿类动物模型NMS 16敏感性，但尽管产后膀胱炎23-25 ​​的长期影响广泛的特性，早期生活压力的影响已经在很大程度上消失在未学泌尿生殖器官。因此，下面的研究将描述如何在小鼠中进行NMS和后来评价perigenital机械灵敏度和肥大细胞浸润/活化在前列腺来验证使用雄性网管小鼠作为CP / CPPS的临床前模型。 所有的诊断慢性盆腔疼痛性疾病的，CP / CPPS可能是最公认和特点综合症，尽管有大约14％的26终生患病率和估计4400美元（每年门诊费用两倍，腰痛或类风湿关节炎27）。患者在会阴，直肠，前列腺，阴茎，睾丸，和/或腹部28，遇到一个喜CP / CPPS报告疼痛gher程度的心理压力比对照组病人29，和目前常用的有或症状被诊断为伴发慢性盆腔疼痛或情绪障碍5,29-31。复发性感染，漏上皮，神经性炎症，和自身免疫都被推测为CP的电位基本原因/ CPPS 2,32,33，以及肥大细胞活化和脱颗粒34。从人带有CP / CPPS表达前列腺分泌物增加了肥大细胞类胰蛋白酶和神经生长因子（NGF）水平34，以及后来的研究证实，类胰蛋白酶和羧肽酶A（CPA3），肥大细胞活化的标志物，也增加了尿CP / CPPS患者35。为肥大细胞的发生和维护CP / CPPS的潜在作用已经动物研究对这一综合征的一大焦点迄今。最常用的啮齿动物模型用于研究的CP / CPPS是一种实验性自身免疫性前列腺炎（EAP）的模型根通过皮下注射前列腺抗原在完全弗氏佐剂，这导致不同程度的取决于所使用34,36-39物种和株前列腺发炎产生的。肥大细胞浸润和活化/脱粒已被证明能增加以下诱导的EAP 34,35,40的。转基因小鼠缺乏既肥大细胞34或类胰蛋白酶受体PAR2 35不发展前列腺战术敏感度以下EAP，不像野生型小鼠的EAP。虽然这种临床前模型复制了许多人的CP / CPPS的特点，诱导方案并不表示人的条件，其中有一个不同的病因，往往不涉及直接的炎症，感染，或前列腺损伤。 早期生活压力对CP / CPPS在人类发展的影响已经在很大程度上未经调查去了哪里;然而，一项研究胡等人 41，证明了我ñ谁报的童年身体，情感史，和/或性虐待是显著更容易出现症状提示CP / CPPS的。此外，他们发现，既疼痛和泌尿分数增加患者的身体和情感滥用史。我们已经证实，在雌性C57BL / 6小鼠同网管范例产生阴道过敏和异常的基因表达在两个阴道和膀胱暗示功能失调HPA轴输出16。这方面的证据结合CP / CPPS患者与其他合并症42，其中IC / PBS和情绪障碍，已经更清楚地显示出早期生活应激暴露12-14链接呈现的高患病率，提供了理由使用网管模型小鼠研究CP / CPPS。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Pregnant C57BL/6 female mice&#160;</td>
			<td>Charles River</td>
			<td>027</td>
			<td>Timed or untimed pregnant females should be monitored daily for in-house birth.</td>
		</tr>
		<tr>
			<td>2L glass beaker(s)</td>
			<td>Sigma-Aldrich</td>
			<td>CLS10002L</td>
			<td>Each NMS litter will be held in the same beaker throughout the 21 day separation period.</td>
		</tr>
		<tr>
			<td>VWR Forced Air Incubator, Basic</td>
			<td>VWR International</td>
			<td>414005-124</td>
			<td>The incubator should be held at 33&#176;C and 50% humidity.</td>
		</tr>
		<tr>
			<td>Touch Test Sensory Evaluator, Kit of 20 (Semmes-Weinstein Von Frey Aesthesiometer for touch assessment)</td>
			<td>Stoelting</td>
			<td>58011</td>
			<td>The following monofilaments should be used for perigenital sensitivity assessment: 1.65 g, 2.36 g, 2.83 g, 3.22 g, 3.61 g, 4.08 g, 4.31 g, and 4.74 g.</td>
		</tr>
		<tr>
			<td>Animal Enclosure (12 mice 6 rats)</td>
			<td>IITC</td>
			<td>435</td>
			<td>Be sure to place a heavy object on top of the individual, acrylic animal enclosures to prevent mice from escaping.&#160;</td>
		</tr>
		<tr>
			<td>Mesh Stand for mice and rats (12 mice 6 rats)</td>
			<td>IITC</td>
			<td>410</td>
			<td>Place stand on a stable table top for comfortable access.</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Sigma-Aldrich</td>
			<td>P5493</td>
			<td>Dilute the 10x PBS stock to 1x PBS.</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td>A 4% paraformaldehyde solution is needed to intracardially perfuse mice and postfix tissue in preparation for mast cell staining.</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S5016</td>
			<td>Cryoprotect fixed tissue in 30% sucrose solution at 4&#176;C overnight.</td>
		</tr>
		<tr>
			<td>Heptane</td>
			<td>Sigma-Aldrich</td>
			<td>246654</td>
			<td>Chill heptane on dry ice to freeze tissue.</td>
		</tr>
		<tr>
			<td>Standard Cryomold&#160;</td>
			<td>VWR International</td>
			<td>4557</td>
			<td>To assist in freezing prostate tissue prior to cryostat sectioning</td>
		</tr>
		<tr>
			<td>Tissue-Tek OCT Compound</td>
			<td>Sakura</td>
			<td>4583</td>
			<td>12 x 125 mL</td>
		</tr>
		<tr>
			<td>VWR Superfrost Plus Micro Slide</td>
			<td>VWR International</td>
			<td>48311-703</td>
			<td>75 x 25 x 1 mm</td>
		</tr>
		<tr>
			<td>Toluidine Blue O</td>
			<td>Sigma-Aldrich</td>
			<td>198161</td>
			<td>Certified by the Biological Stain Commission. Suitable for use as a metachromatic stain for mast cells.</td>
		</tr>
		<tr>
			<td>Ethanol, Absolute (200 Proof)</td>
			<td>Fisher Scientific</td>
			<td>BP2818-4</td>
			<td>Molecular Biology Grade, Fisher Bioreagents; 95% and 100% solutions will be needed to dehydrate stained cryosections before mast cell visualization.</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S9888</td>
			<td>Acidified NaCl solution should be made fresh before staining cryosections.</td>
		</tr>
		<tr>
			<td>GeneMate Sterile 50 mL Centrifuge Tubes&#160;</td>
			<td>BioExpress</td>
			<td>C-3394</td>
			<td>Disposable tubes used to mix toulidine blue elements or dip slides into 1xPBS.</td>
		</tr>
		<tr>
			<td>Coplin staining dish for 10 slides, with ground glass cover</td>
			<td>Fisher Scientific</td>
			<td>08-815</td>
			<td>Hold up to 10 standard 3 x 1 in. (75 x 25mm) slides back-to-back. Use separate dishes for Toluidine Blue working solution, 95% EtOH, 100% EtOH, and xylene.</td>
		</tr>
		<tr>
			<td>Xylenes (Histological)</td>
			<td>Fisher Scientific</td>
			<td>X3P</td>
			<td>Once dehydrated, tissue is cleared with xylene.</td>
		</tr>
		<tr>
			<td>Microscope Slide Boxes, 100-Place</td>
			<td>VWR International</td>
			<td>82003</td>
			<td>Boxes can be used for storage and transportation of slides.</td>
		</tr>
		<tr>
			<td>Fisherbrand Microscope Slide Box</td>
			<td>Fisher Scientific</td>
			<td>22-363-400</td>
			<td>These slide boxes are typically small enough to fit inside a cryostate.</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
			<td>Glycerol is a traditional mounting medium.</td>
		</tr>
		<tr>
			<td>Mounting Medium, Richard-Allan Scientific</td>
			<td>VWR International</td>
			<td>4111</td>
			<td>Mounting medium firmly bonds the coverslip to the slide.</td>
		</tr>
		<tr>
			<td>Micro Cover Glasses, Rectangular, No. 1</td>
			<td>VWR International</td>
			<td>48393-106</td>
			<td>A coverslip is placed over the dehydrated and cleared tissue to protect the sample.</td>
		</tr>
		<tr>
			<td>Light Microscope</td>
			<td>Nikon</td>
			<td></td>
			<td>The Advanced Automated Research Microscope Eclipse 90i, used by our lab, has been discontinued and replaced Eclipse Ni-E.&#160;&#160;</td>
		</tr>
	</tbody>
</table>

assessment of perigenital sensitivity and prostatic mast cell activation in a mouse model of neonatal maternal separation

慢性前列腺炎/慢性骨盆疼痛综合征（CP / CPPS）有14％的终生患病，是50岁以下的泌尿外科最常见的诊断为男性，但它是最不被理解和研究慢性盆腔疼痛症。慢性盆腔疼痛报告显著子在经历了早期的生活压力或滥用，它可以显着地影响到下丘脑 - 垂体 - 肾上腺轴（HPA）的运作和监管。肥大细胞活化，这已被证明是增加两个尿中并表达的CP / CPPS患者的前列腺分泌物，部分是由HPA轴的下游激活调节。新生儿母婴分离（NMS）已使用了二十多年研究的早期生活压力的结果在啮齿类动物模型，包括改变HPA轴和内脏敏感性。在这里，我们提供使用网管作为CP / CPPS的雄性C57BL / 6小鼠的临床前模型中的详细协议。我们描述的方法用于执行网管，评估perigenital机械异常性疼痛，和肥大细胞活化的组织学证据。我们还提供证据表明，早期的心理压力可以让男性泌尿生殖系统的小鼠长期影响。

小鼠经历NMS显示指示CP / CPPS的行为证据。当具有渐变一系列的von Frey单丝8周龄网管小鼠中测试时相比幼稚同行（n = 4时）显示perigenital机械异常性疼痛（5例， 图2）。这是证明作为显著减少（P &lt;0.01;学生t检验）在引起了积极的行为反应的机械痛阈，记录为轻快加加速度或跳跃响应于单丝应用或舔或咬行为朝向单丝。 这暴露于NMS小鼠也显示CP / CPPS的组织学?...

Watch this Scientific Journal Video about Assessment of Perigenital Sensitivity and Prostatic Mast Cell Activation in a Mouse Model of Neonatal Maternal Separation at JoVE.com

Assessment of Perigenital Sensitivity and Prostatic Mast Cell Activation in a Mouse Model of Neonatal Maternal Separation

我们正在测量perigenital机械灵敏度和肥大细胞活化在雄性C57BL / 6小鼠中进行的早期生活应激范式的前列腺 - 以诱导的慢性前列腺炎/慢性骨盆疼痛综合征的临床前模型新生儿母婴分离，。

的Perigenital敏感性和前列腺肥大细胞活化新生母婴分离的小鼠模型评估

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) has a lifetime prevalence of 14% and is the most common urological diagnosis for men under the ...

assessment-perigenital-sensitivity-prostatic-mast-cell-activation

Research

JoVE Journal

Medicine

9.2K 次观看.  University of Kansas Medical Center. 我们正在测量perigenital机械灵敏度和肥大细胞活化在雄性C57BL / 6小鼠中进行的早期生活应激范式的前列腺 - 以诱导的慢性前列腺炎/慢性骨盆疼痛综合征的临床前模型新生儿母婴分离，。 

视频: Assessment of Perigenital Sensitivity and Prostatic Mast Cell Activation in a Mouse Model of Neonatal Maternal Separation

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

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 ...

A Swine Model of Neonatal Asphyxia

Annually more than 1 million neonates die worldwide as related to asphyxia. Asphyxiated neonates commonly have multi-organ failure including hypotension, perfusion deficit, hypoxic-ischemic encephalopathy, pulmonary hypertension, vasculopathic enterocolitis, renal failure and thrombo-embolic complications. Animal models are developed to help us understand the patho-physiology and pharmacology of neonatal asphyxia. In comparison to rodents and newborn lambs, the newborn piglet has been proven to be a valuable model. The newborn piglet has several advantages including similar development as that of 36-38 weeks human fetus with comparable body systems, large body size (&tilde;1.5-2 kg at birth) that allows the instrumentation and monitoring of the animal and controls the confounding variables of hypoxia and hemodynamic derangements.
We here describe an experimental protocol to simulate neonatal asphyxia and allow us to examine the systemic and regional hemodynamic changes during the asphyxiating and reoxygenation process as well as the respective effects of interventions. Further, the model has the advantage of studying multi-organ failure or dysfunction simultaneously and the interaction with various body systems. The experimental model is a non-survival procedure that involves the surgical instrumentation of newborn piglets (1-3 day-old and 1.5-2.5 kg weight, mixed breed) to allow the establishment of mechanical ventilation, vascular (arterial and central venous) access and the placement of catheters and flow probes (Transonic Inc.) for the continuously monitoring of intra-vascular pressure and blood flow across different arteries including main pulmonary, common carotid, superior mesenteric and left renal arteries. Using these surgically instrumented piglets, after stabilization for 30-60 minutes as defined by Z&lt;10% variation in hemodynamic parameters and normal blood gases, we commence an experimental protocol of severe hypoxemia which is induced via normocapnic alveolar hypoxia. The piglet is ventilated with 10-15% oxygen by increasing the inhaled concentration of nitrogen gas for 2h, aiming for arterial oxygen saturations of 30-40%. This degree of hypoxemia will produce clinical asphyxia with severe metabolic acidosis, systemic hypotension and cardiogenic shock with hypoperfusion to vital organs. The hypoxia is followed by reoxygenation with 100% oxygen for 0.5h and then 21% oxygen for 3.5h. Pharmacologic interventions can be introduced in due course and their effects investigated in a blinded, block-randomized fashion.

Large animal models have good translational values in the examination of physiology and pharmacology of neonatal asphyxia. Using newborn piglets, we develop an experimental protocol to simulate neonatal asphyxia which has advantages of studying the systemic and regional hemodynamics, oxygen transport with biochemical and pathologic pathways and correlations.

新生儿窒息了猪模型

Annually more than 1 million neonates die worldwide as related to asphyxia. Asphyxiated neonates commonly have multi-organ failure including hypotension, ...

A Piglet Model of Neonatal Hypoxic-Ischemic Encephalopathy

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the world&#8217;s 2.9 million neonatal deaths. Animal models of HIE have contributed to the understanding of the pathophysiology in HIE, and have highlighted the dynamic process that occur in brain injury due to perinatal asphyxia. Thus, animal studies have suggested a time-window for post-insult treatment strategies. Hypothermia has been tested as a treatment for HIE in pdiglet models and subsequently proven effective in clinical trials. Variations of the model have been applied in the study of adjunctive neuroprotective methods and piglet studies of xenon and melatonin have led to clinical phase I and II trials1,2. The piglet HIE model is further used for neonatal resuscitation- and hemodynamic studies as well as in investigations of cerebral hypoxia on a cellular level. However, it is a technically challenging model and variations in the protocol may result in either too mild or too severe brain injury. In this article, we demonstrate the technical procedures necessary for establishing a stable piglet model of neonatal HIE. First, the newborn piglet (&#60; 24 hr old, median weight 1500 g) is anesthetized, intubated, and monitored in a setup comparable to that found in a neonatal intensive care unit. Global hypoxia-ischemia is induced by lowering the inspiratory oxygen fraction to achieve global hypoxia, ischemia through hypotension and a flat trace amplitude integrated EEG (aEEG) indicative of cerebral hypoxia. Survival is promoted by adjusting oxygenation according to the aEEG response and blood pressure. Brain injury is quantified by histopathology and magnetic resonance imaging after 72 hr.

Hypoxic-ischemic encephalopathy following perinatal asphyxia can be studied using animal models. We demonstrate the procedures necessary for establishing a piglet model of neonatal hypoxic-ischemic encephalopathy.

新生儿缺氧缺血性脑病的小猪模型

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the ...

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

新生儿小鼠脊髓压迫损伤模型

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...

A Battery of Motor Tests in a Neonatal Mouse Model of Cerebral Palsy

As the sheer number of transgenic mice strains grow and rodent models of pediatric disease increase, there is an expanding need for a comprehensive, standardized battery of neonatal mouse motor tests. These tests can validate injury or disease models, determine treatment efficacy and/or assess motor behaviors in new transgenic strains. This paper presents a series of neonatal motor tests to evaluate general motor function, including ambulation, hindlimb foot angle, surface righting, negative geotaxis, front- and hindlimb suspension, grasping reflex, four limb grip strength and cliff aversion. Mice between the ages of post-natal day 2 to 14 can be used. In addition, these tests can be used for a wide range of neurological and neuromuscular pathologies, including cerebral palsy, hypoxic-ischemic encephalopathy, traumatic brain injury, spinal cord injury, neurodegenerative diseases, and neuromuscular disorders. These tests can also be used to determine the effects of pharmacological agents, as well as other types of therapeutic interventions. In this paper, motor deficits were evaluated in a novel neonatal mouse model of cerebral palsy that combines hypoxia, ischemia and inflammation. Forty-eight hours after injury, five tests out of the nine showed significant motor deficits: ambulation, hindlimb angle, hindlimb suspension, four limb grip strength, and grasping reflex. These tests revealed weakness in the hindlimbs, as well as fine motor skills such as grasping, which are similar to the motor deficits seen in human cerebral palsy patients.

Presented is a concise battery of mouse neonatal motor tests. Using these tests, neonatal motor deficits can be demonstrated in a variety of neonatal motor disorders. By having a standardized set of tests, results from different studies can be compared, allowing for better and accurate reporting between groups.

汽车测试的脑瘫新生儿小鼠模型电池

As the sheer number of transgenic mice strains grow and rodent models of pediatric disease increase, there is an expanding need for a comprehensive, ...

Myocardial Infarction in Neonatal Mice, A Model of Cardiac Regeneration

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and regeneration, and to define new targets for therapeutics. For decades, models of complete heart regeneration existed in amphibians and fish, but a mammalian counterpart was not available. The recent discovery of a postnatal window during which mice possess regenerative capabilities has led to the establishment of a mammalian model of cardiac regeneration. A surgical model of mammalian cardiac regeneration in the neonatal mouse is presented herein. Briefly, postnatal day 1 (P1) mice are anesthetized by isoflurane and placed on an ice pad to induce hypothermia. After the chest is opened, and the left anterior descending coronary artery (LAD) is visualized, a suture is placed around the LAD to inflict myocardial ischemia in the left ventricle. The surgical procedure takes 10-15 min. Visualizing the coronary artery is crucial for accurate suture placement and reproducibility. Myocardial infarction and cardiac dysfunction are confirmed by triphenyl-tetrazolium chloride (TTC) staining and echocardiography, respectively. Complete regeneration 21 days post myocardial infarction is verified by histology. This protocol can be used to as a tool to elucidate mechanisms of mammalian cardiac regeneration after myocardial infarction.

This protocol describes a highly reproducible model of cardiac regeneration by surgical induction of myocardial infarction in the left ventricle of postnatal day 1 mice. The method involves induction of hypothermic anesthesia and ligation of the left anterior descending coronary artery.

心肌梗死新生小鼠，心肌再生的模型

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and ...

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 ...

A Cryoinjury Model to Study Myocardial Infarction in the Mouse

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we demonstrate a murine cryoinjury infarct model that generates precise infarct sizes with high reproducibility and replicability. In brief, after intubation and sternotomy of the animal, the heart is lifted from the thorax. The probe of a handheld liquid nitrogen delivery system is applied onto the myocardial wall to induce cryoinjury. Impaired ventricular function and electrical conduction can be monitored with echocardiography or optical mapping. Transmural myocardial remodeling of the infarcted area is characterized by collagen deposition and loss of cardiomyocytes. Compared to other models (e.g., LAD-ligation), this model utilizes a handheld liquid nitrogen delivery system to generate more uniform infarct sizes.

This article demonstrates a model to study cardiac remodeling after myocardial cryoinjury in mice.

研究小鼠心肌梗死的低温损伤模型

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we ...

A Neonatal BALB/c Mouse Model of Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight infants, with high mortality and unclear pathogenesis. The cause of NEC may be related to inflammatory immune regulatory system abnormalities. An NEC animal model is an indispensable tool for NEC disease immune research. NEC animal models usually use C57BL/6J neonatal mice; BALB/c neonatal mice are rarely used. Related studies have shown that when mice are infected, Th2 cell differentiation is predominant in BALB/c mice compared to C57BL/6J mice. Studies have suggested that the occurrence and development of NEC are associated with an increase in T helper type 2 (Th2) cells and are generally accompanied by infection. Therefore, this study used neonatal BALB/c mice to induce an NEC model with similar clinical characteristics and intestinal pathological changes as those observed in children with NEC. Further study is warranted to determine whether this animal model could be used to study Th2 cell responses in NEC.

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight infants, with high mortality and unclear pathogenesis. The cause of NEC may be related to inflammatory immune regulatory system abnormalities. An NEC animal model is an indispensable tool for NEC disease immune research. NEC animal models usually use C57BL/6J neonatal mice; BALB/c neonatal mice are rarely used. Related studies have shown that when mice are infected, Th2 cell differentiation is predominant in BALB/c mice compared to C57BL/6J mice. Studies have suggested that the occurrence and development of NEC are associated with an increase in T helper type 2 (Th2) cells and are generally accompanied by infection. Therefore, this study used neonatal BALB/c mice to induce an NEC model with similar clinical characteristics and intestinal pathological changes as those observed in children with NEC. Further study is warranted to determine whether this animal model could be used to study Th2 cell responses in NEC.

一种新生儿BALB/c坏死性小肠结肠炎小鼠模型

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight ...