The authors would like to thank all members of the Colucci lab for helpful discussions. This work was funded by The Wellcome Trust [094073/Z/10/Z], The Centre for Trophoblast Research and The British Heart Foundation.

在这个手稿中讨论的所有程序都是按照英国内政部法规和剑桥伦理审查委员会的批准。 1.标本采集<ol><li>设置使用8-12周龄的雌性C57BL / 6小鼠成年男性（最多2个每个女性的男性），下午定时交配。检查阴道塞的存在，作为交配的标志在随后的日子里的清晨。塞在早上马克gd0.5的存在。 </li><li>上gd9.5，安乐死动物通过颈脱位和打开腹腔。需要注意的是安乐死的CO 2可能会引起血管扩张。 </li><li>使用牙线轻轻结扎子宫动脉（图1，虚线箭头）。扎周围动脉牙线在每个子宫角，近端至卵巢的尖端，以及围绕子宫颈。 注意:不要破裂的血管，因为这可能会导致崩溃的动脉。 </li> &lt;利&gt;迅速解剖子宫使用解剖剪刀，远侧切除整个子宫的三个连接点在子宫角和子宫颈的尖端。 </li><li>将子宫，对准备好的聚苯乙烯片使两个角都沿着长轴从子宫颈相反的方向对齐。轻轻地伸展并销插入使用2-3个地下25针的地方。 </li><li>浸入聚苯乙烯成制备50ml锥形管中。补足至50ml，用10％福尔马林中。定为5-6小时，在室温下进行。 </li><li>丢弃福尔马林溶液和替换用50ml 1×磷酸盐缓冲盐水（PBS）5分钟。 </li><li>近侧到各端的连接点和近侧切除两个子宫角，以在中心子宫颈，修剪掉周围的植入位置的任何多余的脂肪和在PBS中清洗两次，每次5分钟，以去除福尔马林的痕迹。 </li><li>存储在70％乙醇的样品在4℃（长达两周）直到加工。 注意:的Ethanol是高度易燃的。 </li><li>使用使用表1所示的参数的自动处理系统。在石蜡单独嵌入的角，以便它们可以沿垂直于子宫的长轴进行切割。这确保了每个植入部位的最大面积将在块（图2A）的中间被暴露以供分析。 可选:这是针对该块的随机化方便的时间点，如果需要的。 </li><li>用切片机，切断从块7微米厚的连续切片。在染色行HE每49微米间隔一个部分（H＆E，见2.1）。存储的剩余部分在室温下的SMA染色和其他下游应用。 </li></ol><table fo:keep-together.within-page="1"><tbody><tr height="21"><td height="21" style="height:21px;width:99px;"> 解 </td><td style="width:43px;"> 时间<html></td><td style="width:97px;"> 温度 </td></tr><tr height="21"><td height="21" style="height:21px;"> 70％乙醇</td><td> 1小时</td><td> 40℃ </td></tr><tr height="21"><td height="21" style="height:21px;"> 100％乙醇</td><td> 1小时</td><td> 40℃ </td></tr><tr height="21"><td height="21" style="height:21px;"> 100％乙醇</td><td> 1小时</td><td> 40℃ </td></tr><tr height="21"><td height="21" style="height:21px;"> 100％乙醇</td><td> 1小时</td><td> 40℃ </td></tr><tr height="21"><td height="21" style="height:21px;"> 100％乙醇</td><td> 1小时</td><td> 40℃ </td></tr><tr height="21"><td height="21" style="height:21px;"> 100％乙醇</td><td> 1小时</td><td> 40℃ </td></tr><tr height="21"><td height="21" style="height:21px;"> 100％乙醇</td><td> 1小时</td><td> 40℃ </td></tr><tr height="21">:21px;"&gt;二甲苯</td><td> 1小时</td><td> 40℃ </td></tr><tr height="21"><td height="21" style="height:21px;">二甲苯</td><td> 1小时</td><td> 40℃ </td></tr><tr height="21"><td height="21" style="height:21px;">二甲苯</td><td> 1小时</td><td> 40℃ </td></tr><tr height="21"><td height="21" style="height:21px;">石蜡</td><td> 1小时</td><td> 63℃ </td></tr><tr height="21"><td height="21" style="height:21px;">石蜡</td><td> 1小时</td><td> 63℃ </td></tr><tr height="21"><td height="21" style="height:21px;">石蜡</td><td> 1小时</td><td> 63℃ </td></tr><tr height="22"><td height="22" style="height:22px;">石蜡</td><td> 1小时</td><td> 63℃ </td></tr></tbody></table> 表1:设置用于子宫组织的处理设置用于自动组织处理概述 2. STEReological评估<ol><li> H＆E染色<ol><li> Deparaffinize切片浸入滑动地安装在滑动机架的100％二甲苯浴中10分钟，在室温下。 注意:二甲苯是易燃品，可疑致癌物是通过接触和吸入有毒。这项工作要在通风橱中进行，使用个人防护装备（PPE）。 </li><li>浸入含有清洁二甲苯另外10分钟，在滑动浴。 </li><li>顺序地浸入载玻片在含有100％的乙醇，95％乙醇，70％乙醇与纯净水，每次5分钟浴。 </li><li>沉浸载玻片于50％吉尔否3苏木溶液，用纯化水稀释，5分钟。 注意:苏木是通过摄入和接触对眼睛有害。在处理时应佩戴合适的个人防护装备。 </li><li>流动的自来水10分钟在染色槽洗幻灯片。 </li><li>在1％的浸入滑动酸/醇溶液（1％10M的盐酸在70％乙醇），持续10秒和洗涤，并在染色槽流动的自来水。 </li><li>浸泡在曙红溶液玻片30秒和下流动的自来水10分钟在染色槽清洗。 </li><li>内通风橱，依次浸入载玻片在含有70％乙醇，95％乙醇和100％乙醇，每次5分钟浴。 </li><li>浸入含有二甲苯10分钟洗澡幻灯片。 </li><li>摩盖玻片用二甲苯基的安装平台。在之前的可视化，在显微镜下将载玻片在通风橱干燥水平地和彻底。 </li></ol></li><li>血管的大小和重塑状态体视评估<ol><li>对于每个着床部位，确定哪些部分是靠近正中矢状点。胎儿和胎盘显影之间的绒毛尿囊附着用作正中矢状点的良好指标。 </li><li>选择3个部分，在49微米的间隔接近正中矢状点进行分析。扫描选定的幻灯片。为了避免增量泸静脉，这已被证明是更位于边缘的在植入部位16，限制在每个植入位点（图2A）的中央2/4的分析。 </li><li>为了评估管腔大小，绘制血管周围的内侧，并记录此形状的面积。总容器大小，绘制围绕容器的外壁上，包括内皮细胞，周细胞和壁间白细胞。 注意:总血管大小的腔比为容器的重塑状态的代理。作为船只通过该部被切断沿平面线圈，可能存在在一个滑动同一动脉的多个横截面。避免测量同一动脉多次。 </li><li>记录在每个种植部位与最大和最圆流明的5艘测量。测量一式三份的每个植入位点（三个部分隔开49微米彼此分开）。这些15个测量represe平均NTS的最大船只的平均直径。 </li><li>为了避免一些样品中较大的血管比例过高，测量容器中的每个植入位点的数目相同。 </li></ol></li></ol> 3.定性评估采用免疫组化<ol><li>脱蜡和补液<ol><li> Deparaffinize切片浸入滑动安装在耐热滑架的100％二甲苯浴，在室温下10分钟。 </li><li>浸入含有清洁二甲苯另外10分钟，在滑动浴。 </li><li>顺序地浸入载玻片在含有100％的乙醇，95％乙醇和70％，纯化水，每次5分钟浴。 </li></ol></li><li>抗原检索<ol><li>制备在纯水中的10 mM柠檬酸钠缓冲液，并调整至pH 6，用10摩尔的盐酸。 </li><li>填写一个耐热的容器，可以容纳含有足够笏国内压力锅600毫升用400毫升柠檬酸钠缓冲液和地点呃淹没容器的一半。松散地连接高压锅热的盖，直至沸腾。 </li><li>地方玻片架放入柠檬酸钠缓冲液和紧密固定压力锅的盖上。一旦加压，使沸腾3分钟。 </li><li>降压压力锅，取出内部容器。允许滑动到在柠檬酸钠缓冲液冷却，在室温下10分钟。 </li><li>洗幻灯片中含有纯净水5分钟洗澡。 </li><li>环绕使用疏水性屏障笔的部分。 </li><li>通过浸没在PBS 5分钟洗幻灯片。 </li></ol></li><li>阻断内源性过氧化物酶和非特异性结合<ol><li>孵育3％过氧化氢稀释在纯水中，在湿润的腔室30分钟，在室温下部分。 注意:过氧化氢是强烈刺激眼睛，皮肤和在摄入。穿戴合适的个人防护装备。 </li><li>挖掘掉多余的过氧化氢溶液和洗涤SLI德两次通过浸没在Tris缓冲盐水（TBS）中，每次2分钟。 </li><li>孵育小鼠免疫球蛋白封闭试剂从根据制造商的说明制备对小鼠套件鼠标部分。 </li><li>洗涤滑动两次在TBS中每2分钟。 </li></ol></li><li>平滑肌肌动蛋白免疫检测<ol><li>制备抗体稀释液（在试剂盒中提供），并孵育切片5分钟，在室温下，或根据制造商的说明。 </li><li>稀释1:100的小鼠抗人平滑肌肌动蛋白（DAKO M0851）在抗体稀释液溶液。稀释在抗体稀释溶液小鼠IgG2a同种型对照，以确保最终的免疫球蛋白浓度在两种解决方案等效的。 </li><li>点击多余的抗体稀释溶液，并覆盖部分与任何稀释的抗体或同种型控制解决方案。孵育在一个加湿室中30分钟，在室温下进行。 </li><li>洗涤滑动两次在TBS中每2分钟。 </li><li>稀释生物素化的抗小鼠二抗孵育切片在室温下10分钟，或根据制造商的说明。 </li><li>洗涤滑动一旦各用TBS和PBS进行2分钟，分别。 </li><li>根据制造商的说明制备3,3&#39;-二氨基联苯胺（DAB）溶液。顶盖采用DAB溶液部分和孵化长达4分钟，确保监测部分，以避免过多的颜色变化。 注意:DAB是易燃品，可疑致癌物是通过接触和吸入有毒。在处理时应佩戴合适的个人防护装备。 </li><li>沉浸在纯净水滑梯，一旦染色所需的强度达到。 </li><li>染液用50％吉尔号3苏木为20秒，并在流动的自来水直到水运行清楚在染色槽清洗。 </li></ol></li><li>脱水和安装<ol><li>在通风橱中，依次浸泡在含70％ETH浴场幻灯片anol，95％乙醇和100％乙醇，每次5分钟。 </li><li>浸入含有二甲苯10分钟洗澡幻灯片。 </li><li>摩盖玻片用二甲苯基的安装平台。在通风橱中可视化，在显微镜下将载玻片事先干燥水平地和彻底地</li></ol></li></ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+Ultrasonography+to+Define+Fetal-Maternal+Relationships:+Moving+from+Humans+to+Mice.">Using Ultrasonography to Define Fetal-Maternal Relationships: Moving from Humans to Mice.</a> Comparative Med. 59 (6), 527-533 (2009).</li></ol>

The authors have nothing to disclose.

这里介绍的协议的目的是提供以用来评估所必需的成功怀孕的血管变化可再现方法。临界本评估的成功是组织切片的质量。这两个精确确定的植入部位的孕周，以及可靠的结扎子宫动脉是关键步骤。变化的参数，如时间固定的，组织处理协议等等也可能影响结果。对于无偏体视学评估，以测量在每个植入部位血管的数目相同是很重要的。它建议以测量每植入部位并且每个植入位点以一式三份5船只。这15个测量值的平均值代表着5大血管平均尺寸。 另外，从alymphoid 的Rag2数据- / - IL2RG - / -这里显示的小鼠，我们发现这PROTocol为一些的母体免疫功能不足的模型，其中包括抑制NK细胞14的一个很有用的。在动脉重塑缺陷也被证明在受损滋养细胞侵袭18的模型和此处所描述的技术可能会有所帮助，以评估在这些情况下，脉管。我们优化和验证此协议为蜕膜血管重塑在妊娠中期在小鼠调查，但还可以想到，以适应这个协议在其他模型系统 （例如大鼠）或甚至在其他器官中工作。而我们发现，对NK依赖性重塑可以鲁棒地评估在gd9.5，它可修正为其它时间点，如gd12.5，当产妇脉管被完全改造。在这个时间点，血管内滋养细胞浸润是更深16和这个时间点可以是更适合于滋养层的血管变化的作用的调查。如果额外的量化读数的需要，研究者还可以增加染色的SMA部的数量和每个植入位点内的评估部分改造血管的百分比。 该协议的限制是组织学检查的描述性。功能测定，然而，仅慢慢变得可用（例如多普勒超声19）。这些技术需要专业技术知识和用于获取和维护设备更高的费用，但是具有提供在体内的读出纵向为，可以在组织学可观察到的变化的影响的优点。所有的体视学考试的一个可能的缺点是研究者的主观性。为此，使用分析所有样品独立两个独立的考官可以帮助避免这个问题。而这里所概述的协议提供了洞察多少血如何到达胎母接口，它可能是有利的，可以与并发症结合通过塑料评估血液的总量在任何给定时间在脉管的ementary方法蒙上16。 该协议的优点是，它结合了两个独立的方法。血管变化是首次定量体视学评估，然后将结果进行验证定性采用免疫组化。结果的可靠性可以通过随机样品得到进一步加强。此协议中制备的样品可以容易地用于还研究怀孕的其他参数，如decidualisation，妊娠（MLAP）或感兴趣的细胞的免疫检测的mesometrial淋巴聚集体的发展，在妊娠中期到严格评估怀孕的表型。 动脉重构是妇女和这些变化的失败简单怀孕的关键一步巩固了很大的产科综合征（GOS），即先兆子痫，胎儿生长受限死胎。我们在座的技术来仔细评估，模仿GOS的病理生理的一些特点小鼠模型中怀孕的表型。

营养物质，气体和废产物的过程中真哺乳亚纲妊娠交换是由胎盘介导的。在女性生殖道，子宫动脉是血液的主要管道到子宫。植入后，这些分支成为专门的容器被称为螺旋动脉通过底蜕膜对胎儿胎盘单位线圈。直径和这些螺旋动脉，这是由白细胞包围，尤其子宫自然杀伤（UNK）细胞的弹性，决定体积和血液可用速度胎盘1,2。正确的血流动力学改变为螺旋动脉重塑的结果是妊娠成功的关键。 而潜在的机制在人和鼠妊娠之间的细节不同，在这两个物种重塑过程的最终结果是扩张型，高电导脉管即失去了它的平滑肌层。在小鼠中，UNK细胞衍生的干扰素（IFN-）γ就要引起这些变化在大约妊娠中期3-5。缺乏或者NK细胞或IFN-γ信号传导途径的组分小鼠不能经受这些变化，这是与减少胎儿生长6,7-关联，强调的充足的血液供应，胎儿胎盘单元的重要性。在人类早期妊娠，外滋养细胞的间质及血管内入侵及其与UNK细胞的相互作用是引起血管改变，促进胎儿生长8-10重要。 UNK细胞可以通过趋化因子11和细胞因子如粒细胞巨噬编排人类滋养细胞浸润-集落刺激因子（GM-CSF）12。浅滋养细胞浸润与减少动脉重塑和妊娠并发症，如先兆子痫9相关联。 该协议是基于安妮·克罗伊是谁介绍的很重要的开创性工作免疫系统，特别是NK-衍生的IFN-γ通过免疫组织化学和优雅转移实验5,13成功血管重塑的作用。在这里，我们描述了一种快速而经济的方式，以评估螺旋动脉重塑状态。虽然我们实验室常规于妊娠中期（妊娠天（GD）9.5）14评估这个，重塑过程需要gd8.5和12.5 15之间的地方。自动幻灯片扫描仪的出现，极大地促进了血管和管腔面积的评估由部分，我们发现这是不是测量血管直径更可重复的方法。在加入免疫组化检测的SMA的允许从体视评估获得的结果的一个简单的验证。如同所有的体视技术，建议由至少两个审查员来执行评估作为一个盲实验。为此目的，一个随机化步骤可以引入时串行地切婷的样本，以便评估的幻灯片研究者不知道从哪个实验组的样品来。 该协议的总体目标是提供一个可靠的工具来仔细评估螺旋动脉重塑在小鼠中定义母亲和父亲的基因型，在小鼠模型妊娠并发症的范围内。结果强调在此过程中，这是正常的胎儿生长所必需的细胞UNK的依赖性​​。

<table border="0" cellpadding="0" cellspacing="0" width="704">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:212px;">C57BL/6 mice (8-12 weeks old)</td>
			<td style="width:99px;">Charles River</td>
			<td style="width:111px;"></td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Dental floss</td>
			<td style="width:99px;"></td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Polystyrene</td>
			<td style="width:99px;"></td>
			<td style="width:111px;"></td>
			<td>Used for holding tissue in place</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Straight dissection scissors</td>
			<td style="width:99px;">EMS</td>
			<td style="width:111px;">72940</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">25G needles</td>
			<td style="width:99px;">BD&#160;</td>
			<td style="width:111px;">300600</td>
			<td>Used for holding tissue in place</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">50 ml conical tubes</td>
			<td style="width:99px;">Falcon</td>
			<td style="width:111px;">352070</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:212px;">Formalin solution, neutral buffered, 10%</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:111px;">HT501128-4L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Phosphate buffered saline</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:111px;">P3813-10PAK</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Ethanol</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:111px;">32221-2.5L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Paraffin</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:111px;">327204-1KG</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:212px;">Xylene</td>
			<td style="width:99px;">Fisher Scientific</td>
			<td style="width:111px;">X/0250/17</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Automated tissue processor</td>
			<td style="width:99px;">Sakura</td>
			<td style="width:111px;">6032</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Microtome</td>
			<td style="width:99px;">Leica</td>
			<td style="width:111px;">RM2245</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Slide rack</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:111px;">Z710989</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Coplin jar</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:111px;">S5891</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Purified water</td>
			<td style="width:99px;"></td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:212px;">Hematoxylin solution, Gill No. 3</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:111px;">GHS332-1L</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:212px;">Eosin Y solution</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:111px;">HT110116-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Fume hood</td>
			<td style="width:99px;"></td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Xylene-based mounting media</td>
			<td style="width:99px;">VWR</td>
			<td style="width:111px;">361254D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Slide scanner</td>
			<td style="width:99px;">Hamamatsu</td>
			<td style="width:111px;">NanoZoomer</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Slide scanner software</td>
			<td style="width:99px;">Hamamatsu</td>
			<td style="width:111px;">NDP viewer</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:212px;">Sodium citrate tribasic dihydrate</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:111px;">32320</td>
			<td>Make 10 mM solution and adjust pH to 6</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Pressure cooker</td>
			<td style="width:99px;"></td>
			<td style="width:111px;"></td>
			<td>Used for antigen retrieval</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Heating plate</td>
			<td style="width:99px;"></td>
			<td style="width:111px;"></td>
			<td>Used for antigen retrieval</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Hydrophobic barrier pen</td>
			<td style="width:99px;">Vector</td>
			<td style="width:111px;">H-4000</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:212px;">Hydrogen peroxide</td>
			<td style="width:99px;">Fisher Scientific</td>
			<td style="width:111px;">H/1800/15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Tris buffered saline</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:111px;">T6664-10PAK</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Mouse on mouse kit</td>
			<td style="width:99px;">Vector</td>
			<td style="width:111px;">BMK-2202</td>
			<td>Significantly reduces background</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:212px;">Mouse anti-human smooth muscle actin</td>
			<td style="width:99px;">DAKO</td>
			<td style="width:111px;">M0851</td>
			<td>Cross-reacts with mouse</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">IgG2a isotype control</td>
			<td style="width:99px;">DAKO</td>
			<td style="width:111px;">X0943</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Humidified chamber</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:111px;">H6644</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:212px;">3,3&#8217;-Diaminobenzidine solution</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:111px;">D4293-5SET</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:212px;">Hydrochloric acid</td>
			<td style="width:99px;">Fisher Scientific</td>
			<td style="width:111px;">H/1050/PB17</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessment of maternal vascular remodeling during pregnancy in the mouse uterus

The placenta mediates the exchange of factors such as gases and nutrients between mother and fetus and has specific demands for supply of blood from the maternal circulation. The maternal uterine vasculature needs to adapt to this temporary demand and the success of this arterial remodeling process has implications for fetal growth. Cells of the maternal immune system, especially natural killer (NK) cells, play a critical role in this process. Here we describe a method to assess the degree of remodeling of maternal spiral arteries during mouse pregnancy. Hematoxylin and eosin-stained tissue sections are scanned and the size of the vessels analysed. As a complementary validation method, we also present a qualitative assessment for the success of the remodeling process by immunohistochemical detection of smooth muscle actin (SMA), which normally disappears from within the arterial vascular media at mid-gestation. Together, these methods enable determination of an important parameter of the pregnancy phenotype. These results can be combined with other endpoints of mouse pregnancy to provide insight into the mechanisms underlying pregnancy-related complications.

的Rag2 - / - IL2RG - / -小鼠缺乏所有成熟淋巴细胞，包括NK细胞17，它们的子宫动脉不能经受周围妊娠中期-5,14-见于同类系野生型C57BL / 6（WT）小鼠中的血管变化。因为这减少重塑的Rag2的结果- / - IL2RG - / -小鼠显示显著较小腔表面区域，表示整体较小的船只，可显现在H＆E染色的切片和量化stereologically（ 图2）。此外，相对壁厚（容器腔比）在这些小鼠的螺旋动脉增高，提示血液供应减少和较高的血流速度。 采用免疫组化检测SMA的这种量化考核确认。它的特点为野生型小鼠的血管中层内失去大部分的SMA的螺旋动脉通过妊娠中期。如果此NK细胞驱动的过程中不发生，SMA保留在容器的介质，并且可以容易地免疫组织化学检测为周围的血管（图3）的环。 <img alt="图1" src="/files/ftp_upload/53534/53534fig1.jpg" /> 图1:小鼠子宫在gd9.5，示意图示出子宫角，子宫颈（蓝色）的相对位置和主要的子宫动脉（红色）。指示是子宫角（箭头）和结扎点（虚线箭头）的长轴线。用于免疫组织化学的部分被沿着视平面切割。 <img alt="图2" src="/files/ftp_upload/53534/53534fig2.jpg" /> 图2:动脉重塑在妊娠中期的体视学评估（A）为例ASSE鲁米和从野生雌性H＆E染色切片总血管领域的底蜕膜的中央2/4 ssment（由垂直黑划定虚线）在gd9.5。在船只在更高的放大倍率，红色虚线划定总血管区，黄色虚线划定管腔面积。栏= 500微米（左），50微米（右）。子宫的长轴是水平行中该取向。 （B）来自WT（C57BL / 6）和量化的Rag2管腔区域（左面板）和管腔面积的比率，以总血管面积（右图） - / - IL2RG - / - 小鼠（在C57BL / 6背景）。每个数据点代表的15个测量（每部5测量，每植入部位3部分）的平均值。棒表示平均N =从每组4怀孕11-13植入部位; p值从双尾，非配对曼 - 惠特尼检验。 WT:野生型。 <img alT ="图3"SRC ="/文件/ ftp_upload / 53534 / 53534fig3.jpg"/&gt; 图3:在蜕膜免疫组化检测的平滑肌肌动蛋白上的WT（上）和代表性的Rag2 SMA染色- / - IL2RG - / -小鼠 （在C57BL / 6背景下，底部）在gd9.5。栏= 100微米。 WT:野生型。

Watch this Scientific Journal Video about Assessment of Maternal Vascular Remodeling During Pregnancy in the Mouse Uterus at JoVE.com

Assessment of Maternal Vascular Remodeling During Pregnancy in the Mouse Uterus

This protocol describes a technique to assess changes in the maternal vasculature during pregnancy in mice. Using stereological methods, remodeling of the decidual spiral arteries is assessed quantitatively and the results confirmed qualitatively using immunohistochemistry.

产妇血管重构的评估在怀孕期间在小鼠子宫

The placenta mediates the exchange of factors such as gases and nutrients between mother and fetus and has specific demands for supply of blood from the ...

assessment-maternal-vascular-remodeling-during-pregnancy-mouse

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

13.1K 次观看.  University of Cambridge. This protocol describes a technique to assess changes in the maternal vasculature during pregnancy in mice. Using stereological methods, remodeling of the decidual spiral arteries is assessed quantitatively and the results confirmed qualitatively using immunohistochemistry. 

视频: Assessment of Maternal Vascular Remodeling During Pregnancy in the Mouse Uterus

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into the developing oocyte. Because these events rely on maternal products which typically act very soon after fertilization-that preexist inside the egg, standard approaches for expression and functional reduction involving the injection of reagents into the fertilized egg are typically ineffective. Instead, such manipulations must be performed during oogenesis, prior to or during the accumulation of maternal products. This article describes in detail a protocol for the in vitro maturation of immature zebrafish oocytes and their subsequent in vitro fertilization, yielding viable embryos that survive to adulthood. This method allows the functional manipulation of maternal products during oogenesis, such as the expression of products for phenotypic rescue and tagged construct visualization, as well as the reduction of gene function through reverse-genetics agents.

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

An optimized protocol for the in vitro maturation of zebrafish oocytes used for the manipulation of maternal gene products is presented here.

产妇基因产品的官能化操作使用<em&gt;体外</em斑马鱼&gt;卵母细胞成熟

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into ...

科研

发育生物学

Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for studying early embryonic development. In the absence of leukemia inhibitory factor (LIF), ESCs differentiate by default into neural precursor cells. They can be amassed into a three dimensional (3D) spherical aggregate termed embryoid body (EB) due to its similarity to the early stage embryo. EBs can be seeded on fibronectin-coated coverslips, where they expand by growing two dimensional (2D) extensions, or implanted in 3D collagen matrices where they continue growing as spheroids, and differentiate into the three germ layers: endodermal, mesodermal, and ectodermal. The 3D collagen culture mimics the in vivo environment more closely than the 2D EBs. The 2D EB culture facilitates analysis by immunofluorescence and immunoblotting to track differentiation. We have developed a two-step neural differentiation protocol. In the first step, EBs are generated by the hanging-drop technique, and, simultaneously, are induced to differentiate by exposure to retinoic acid (RA). In the second step, neural differentiation proceeds in a 2D or 3D format in the absence of RA.

We describe a technique for using murine embryonic stem cells for generating two or three dimensional embryoid bodies. We then explain how to induce neural differentiation of the embryoid body cells by retinoic acid, and how to analyze their state of differentiation by progenitor cell marker immunofluorescence and immunoblotting.

二维和三维胚体小鼠胚胎干细胞的维甲酸诱导神经分化的分析

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

新型乳腺脂肪移植技术可视化血管内皮干细胞的血管生成

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

小鼠第二腭融合的实时成像

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is routinely used as an in vivo model to study embryo implantation and pregnancy progression. Unfortunately, such murine studies require pregnancy interruption to enable follow-up phenotypic analysis. To address this issue, we used three-dimensional (3-D) reconstruction of HFUS imaging data for early detection and characterization of murine embryo implantation sites and their individual developmental progression in utero. Combining HFUS imaging with 3-D reconstruction and modeling, we were able to accurately quantify embryo implantation site number as well as monitor developmental progression in pregnant C57BL6J/129S mice from 5.5 days post coitus (d.p.c.) through to 9.5 d.p.c. with the use of a transducer. Measurements included: number, location, and volume of implantation sites as well as inter-implantation site spacing; embryo viability was assessed by cardiac activity monitoring. In the immediate post-implantation period (5.5 to 8.5 d.p.c.), 3-D reconstruction of the gravid uterus in both mesh and solid overlay format enabled visual representation of the developing pregnancies within each uterine horn. As genetically engineered mice continue to be used to characterize female reproductive phenotypes derived from uterine dysfunction, this method offers a new approach to detect, quantify, and characterize early implantation events in vivo. This novel use of 3-D HFUS imaging demonstrates the ability to successfully detect, visualize, and characterize embryo-implantation sites during early murine pregnancy in a non-invasive manner. The technology offers a significant improvement over current methods, which rely on the interruption of pregnancies for gross tissue and histopathologic characterization. Here we use a video and text format to describe how to successfully perform ultrasounds of early murine pregnancy to generate reliable and reproducible data with reconstruction of the uterine form in mesh and solid 3-D images.

Mice are widely used to study gestational biology. However, pregnancy termination is required for such studies which precludes longitudinal investigations and necessitates the use of large numbers of animals. Therefore, we describe a non-invasive technique of high-frequency ultrasonography for early detection and monitoring of post-implantation events in the pregnant mouse.

三维高频超声对小鼠早期妊娠特征的新应用

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is ...

Ovarian Tissue Culture to Visualize Phenomena in Mouse Ovary

Mammalian females periodically ovulate an almost constant number of oocytes during each estrus cycle. To sustain such regularity and periodicity, regulation occurs at the hypothalamic-pituitary-gonadal axis level and on developing follicles in the ovary. Despite active studies, follicle development mechanisms are not clear because of the several steps involved from the dormant primordial follicle activation to ovulation, and because of the regulation complexity that differs at each follicular stage. To investigate the mechanisms of follicle development, and the dynamics of follicles throughout the estrus cycle, we developed a mouse ovarian tissue culture model that can be used to observe follicle development using a microscope. Systematic follicle development, periodical ovulation, and follicle atresia can all be reproduced in the cultured ovary model, and the culture conditions can be experimentally modulated. Here, we demonstrate the usefulness of this method in the study of the regulatory mechanisms of follicle development and other ovarian phenomena.

Ovarian tissue cultures can be used as models of follicle development, ovulation, and follicle atresia and indicate regulatory mechanisms of dynamic ovarian processes.

卵巢组织培养对小鼠卵巢现象的可视化

Mammalian females periodically ovulate an almost constant number of oocytes during each estrus cycle. To sustain such regularity and periodicity, ...

Assessment of Ultrastructural Neuroplasticity Parameters After In Utero Transduction of the Developing Mouse Brain and Spinal Cord

The present study combines in utero transduction with transmission electron microscopy (TEM) aiming at a precise morphometrical analysis of ultrastructural parameters in unambiguously identified topographical structures, affected by a protein of interest that is introduced into the organism via viral transfer. This combined approach allows for a smooth transition from macrostructural to ultrastructural identification by following topographical navigation maps in a tissue atlas. High-resolution electron microscopy of the in-utero-transduced tissue reveals the fine ultrastructure of the neuropil and its plasticity parameters, such as cross-sectioned synaptic bouton areas, the number of synaptic vesicles and mitochondria within a bouton profile, the length of synaptic contacts, cross-sectioned axonal areas, the thickness of myelin sheaths, the number of myelin lamellae, and cross-sectioned areas of mitochondria profiles. The analysis of these parameters reveals essential insights into changes of ultrastructural plasticity in the areas of the nervous system that are affected by the viral transfer of the genetic construct. This combined method can not only be used for studying the direct effect of genetically engineered biomolecules and/or drugs on neuronal plasticity but also opens the possibility to study the in utero rescue of neuronal plasticity (e.g., in the context of neurodegenerative diseases).

The combination of transmission electron microscopy and in utero transduction is a powerful approach for studying morphological changes in the fine ultrastructure of the nervous system during development. This combined method allows deep insights into changes in structural details underlying neuroplasticity with respect to their topographical representation.

小鼠脑脊髓子宫转导后超微结构神经可塑性参数的评价

The present study combines in utero transduction with transmission electron microscopy (TEM) aiming at a precise morphometrical analysis of ...

Determining Bile Duct Density in the Mouse Liver

Mouse is broadly used as a model organism to study biliary diseases. To evaluate the development and function of the biliary system, various techniques are used, including serum chemistry, histological analysis, and immunostaining for specific markers. Although these techniques can provide important information about the biliary system, they often do not present a full picture of bile duct (BD) developmental defects across the whole liver. This is in part due to the robust ability of the mouse liver to drain the bile even in animals with significant impairment in biliary development. Here we present a simple method to calculate the average number of BDs associated with each portal vein (PV) in sections covering all lobes of mutant/transgenic mice. In this method, livers are mounted and sectioned in a stereotypic manner to facilitate comparison among various genotypes and experimental conditions. BDs are identified via light microscopy of cytokeratin-stained cholangiocytes, and then counted and divided by the total number of PVs present in liver section. As an example, we show how this method can clearly distinguish between wild-type mice and a mouse model of Alagille syndrome. The method presented here cannot substitute for techniques that visualize the three-dimensional structure of the biliary tree. However, it offers an easy and direct way to quantitatively assess BD development and the degree of ductular reaction formation in mice.

We present a rather simple and sensitive method for accurate quantification of bile duct density in the mouse liver. This method can aid in determining the effects of genetic and environmental modifiers and the effectiveness of potential therapies in mouse models of biliary diseases.

确定小鼠肝脏中的胆汁密度

Mouse is broadly used as a model organism to study biliary diseases. To evaluate the development and function of the biliary system, various techniques ...

Vascular Casting of Adult and Early Postnatal Mouse Lungs for Micro-CT Imaging

Blood vessels form intricate networks in 3-dimensional space. Consequently, it is difficult to visually appreciate how vascular networks interact and behave by observing the surface of a tissue. This method provides a means to visualize the complex 3-dimensional vascular architecture of the lung.
To accomplish this, a catheter is inserted into the pulmonary artery and the vasculature is simultaneously flushed of blood and chemically dilated to limit resistance. Lungs are then inflated through the trachea at a standard pressure and the polymer compound is infused into the vascular bed at a standard flow rate. Once the entire arterial network is filled and allowed to cure, the lung vasculature may be visualized directly or imaged on a micro-CT (&#181;CT) scanner.
When performed successfully, one can appreciate the pulmonary arterial network in mice ranging from early postnatal ages to adults. Additionally, while demonstrated in the pulmonary arterial bed, this method can be applied to any vascular bed with optimized catheter placement and endpoints.

The aim of this technique is ex vivo visualization of pulmonary arterial networks of early postnatal and adult mice through lung inflation and injection of a radio-opaque polymer-based compound via the pulmonary artery. Potential applications for casted tissues are also discussed.

用于微CT成像的成人和早期产后小鼠肺的血管铸造

Blood vessels form intricate networks in 3-dimensional space. Consequently, it is difficult to visually appreciate how vascular networks interact and ...