authors gratefully acknowledge funding support from the Molly Towell Perinatal Research Foundation, Canadian Institutes of Health Research (CIHR), and Fonds de Recherche du Qu&#233;bec &#8211; Sant&#233; (FRQS) (to MGF) and CIHR-Quebec Training Network in Perinatal Research (QTNPR) (to LDD).
The authors wish to thank Esther Simard, Marco Bosa, Carl Bernard and Carmen Movila for technical assistance.

动物保健随后加拿大议会关于动物饲养和批准蒙特利尔大学理事会动物保健（协议＃10言语报 - 1560年）的指导方针。详细资料，使用的材料和方法是表1中提供。 1.麻醉<ol><li>插入一个单腔导管插入颈静脉。 </li><li>稳重的使用母羊乙酰丙嗪（Atravet 10毫克/毫升）2毫克静脉内约30分钟前，麻醉诱导，以减少与该过程从而降低皮质醇水平有关应力。 </li><li>施用西泮（地西泮5毫克/毫升）20毫克，氯胺酮（的Ketalar 100毫克/毫升）4-5毫克/千克，异丙酚（异丙酚10毫克/毫升）0.5至1毫克/公斤静脉内以诱导全身麻醉。 </li><li>插入气道交换导管插入用喉镜与威斯康星型刀片气管（超长350毫米左手刀），帮助插管。滑动硅气管内管（9至12毫米内径）关闭气道交换导管和进气管。该技术有利于气管插管的过程。充气气管导管的套囊小心，以避免气管压力诱导溃疡和固定所述管的母羊的头部。 </li><li>气管导管连接到麻醉机的呼吸回路，并立即开始机械通气。调整呼吸机设置保持P上的 CO 2内的35至45毫米汞柱正常限度。 </li><li>插入导管进入耳动脉（22至20g; 1个在[0.9×25 mm]的在1.16 [1.1×30 mm]的），并连接到不符合要求的管监测直接动脉血压。 </li><li>使用多参数生理监视器来记录心电图，直接动脉血压，氧饱和度（SPO 2），二氧化碳浓度监测仪（P ET的 CO 2），以及温度每5分钟。传输所有的pH通过串行电缆ysiologic数据到中央生理数据收集计算机。使用循环水毯维持正常体温。 </li><li>管理一个平衡的聚离子溶液以10mL / kg的全身麻醉的第一个小时，然后降低至5毫升/千克/小时。 </li><li>辖甲氧苄啶，磺胺5毫克/公斤四母羊之前的皮肤切口抗生素预防。 </li><li>使用标准的无菌技术与母羊和胎儿的所有手术操作。 </li><li>屏障护士母羊在任何时候。这也包括非手术人员。这将最大限度地减少地方性的潜力，例如，贝氏柯克斯体，使用手套和口罩（N-95型），在任何时候。 </li></ol>手术过程2概述<ol><li>使通过下腹壁立即颅到乳房通过白线20厘米中线切口以减少腹部肌肉损伤。 <ol><li> řetract大网膜颅及子宫角的手动触诊。触诊从子宫体各角到喇叭注意到胎儿和它们的大小的数目的尖端。如果有一个以上的胎儿，通过手动评估所述头部大小和轨道之间的宽度选择较大胎儿。 </li><li>坚决通过部分形象化子宫握住选择胎儿的头部。在大曲率与梅岑鲍姆剪刀执行10cm的子宫切开术。立即把在头一个非乳胶无菌手术手套充满无菌生理盐水，好像它是一个手。另外，使用湿四驱车保持胎头湿润。固定子宫到腹壁。 <ol><li> Exteriorize左右胸四肢，轻轻将胎儿从子宫起来的xyphoid过程。 </li></ol></li><li>插入聚乙烯导管插入右和左肱静脉和动脉使用标准切下来的技术。插入anoth呃聚乙烯导管进入羊膜腔通过行了吧其&#39;末端到胎儿的胸骨。 <ol><li>仅使用无菌导尿管。我们建议在标准设备气体灭菌。将每个导管在针和针为双活塞，以允许购买血液采样或压力监控。 </li></ol></li><li>缝合不锈钢电极的柄，剑突以及每个点的肩监视心电图（ECG）的。 </li><li>返回胎儿到子宫。通过一个小刺切口，在左翼所有导管和电极退出。 </li><li>采用三层关闭关闭剖腹切口。用合成的可吸收的单纤维缝合线的USP 2在一个简单的连续方式缝合腹壁。用合成的可吸收缝合线编织的USP 0在一个简单的连续的方式关闭皮下空间。采用手术不锈钢钉，关闭皮肤切口。 </li></ol></li><lI&gt;手术过程的详细描述<ol><li>通过用4％氯己定葡糖酸盐和软毛刷3分钟剃xyphoid过程中对乳腺和沿着所述侧翼的褶皱在每一侧用刀片＃40清洁腹侧腹部然后彻底除去毛。 <ol><li>执行标准的无菌擦洗葡萄糖酸洗必泰4％从腹部的中心开始，前进中的离心方式3分钟。倒在腹部无菌生理盐水以去除消毒肥皂。为手术准备的最后步骤中，执行的洗必泰溶液2％和异丙醇70％3个替代的通路。 </li></ol></li><li>确保麻醉深度是足够的前切口。建立从脐部只是颅给乳房一个标准的剖腹手术切口穿过白线，以减少腹部的肌肉损伤。 </li><li>插入腹部长海绵钳沿左腹壁起来对计划出口部位在paracostal区域的导管。 <ol><li>推靠墙钳提示，直到助理可以找到它，并确定相应的站点。打开略（1厘米）的钳爪并让助手做2厘米全层切开刺。 </li><li> Exteriorize通过切口的钳提示，再次打开，并轻轻抓住导管与该最后拉出腹部通过腹侧的腹部切口的钳子。值得注意的是，一些群体植入导管，然后exteriorize他们。这具有这样的缺点，即intrasurgical胎儿ECG监测是不可能的。 </li></ol></li><li>触诊子宫，以确定胎儿的位置和电话号码。使用耳间距离确定最大胎儿。切开的大曲率在头部的背部子宫壁，避免了子叶。 <ol><li>通过胎盘膜插入一个钝端插管以获得时niotic流体样品无出血。切开用剪刀的胎盘胎膜。 </li></ol></li><li>通过此切口Exteriorize胎儿颅一半。放置一个无菌无乳胶外科手套充满无菌生理盐水，在37℃以上的胎儿头部，以帮助保持正常体温。 <ol><li>在取出胎儿上体从子宫，具有辅助保持巴布科克的，以防止羊水的损失。然后，用钳子巴布科克再次，夹紧胎膜和子宫壁到皮肤以防止腹部污染羊水。 </li><li>暴露胎儿体内的只需要进行检测，并保持在子宫内的剩余部分，​​或者，覆盖潮湿和温暖的（37℃）的无菌布分别的部分。 </li></ol></li><li>两个外展胸肢，以方便接触到的肱动脉和静脉两侧。沿着这两个前臂和护理的内侧面切开全面剖析肱动脉和静脉周围。 </li><li>插入聚乙烯导管插入右和左肱动脉和左肱静脉使用标准切下来的技术。 <ol><li>释放船只从邻近组织插管超过1厘米。用编织合成可吸收的USP 2-0缝合线结扎血管的远端部分。 Preplace一个连字在容器的近端方面，但保持解开。 <ol><li>使用Castroviejo剪，容器横向于它的直径大约为30％。部分地通过拉动近端缝合阻止血液流动。插入导管在近端方向。 </li></ol></li><li>将聚乙烯导管达8厘米近端或直到电阻检测，然后拉回来咯。使用血管钳暂时固定导管的近端的方面。周围放置近端和导管的远侧方面的另一种缝合线。一名助理连续ASPirating和冲洗导管，以确保导管的通畅。 </li><li>关闭使用USP 2-0编织合成可吸收缝合线的胎儿皮肤，用连续缝合模式。 </li></ol></li><li>向右和左肩，柄与xyphoid过程，固定绝缘不锈钢电极，以促进胎儿心电图监测。 </li><li>缝合羊膜压力和取样导管到胸骨。该导管窗孔在其末端。 </li><li>安全上使用一种USP 2-0编织合成的可吸收的缝合材料胎儿的背部所有导管。 </li><li>之前更换胎儿放回子宫，辖克仑特罗30微克四慢慢在15分钟内，以避免低血压和提供子宫松弛。 </li><li>缝合采用USP 4-0编织合成可吸收缝合线材料的连续图案的胎膜。将只有一个导管或电极在同一时间进入CLOSURE保证密封要求。使用双层库欣图案尊重霍尔斯特德原则使用美国药典0编织合成可吸收缝合材料，关闭子宫肌层。仔细埋葬手术结。 </li><li>使用荷包缝合方式，保证所有的导管和心电电缆，因为他们退出离开paracostal切口。 </li><li>使用USP 2单丝合成可吸收缝合材料安全白线与连续图案。关闭使用USP 2-0编织合成可吸收缝合线材料的连续图案的皮下组织。固定皮肤层与手术缝合钉。 </li><li>通过羊膜导管辖250毫克氨苄青霉素静脉滴注，并再次进入羊膜腔。补充丢失的羊水用温生理盐水。 </li><li>将所有的外置导管和心电电极到一个地方袋子保持无菌。周围放置母羊的躯干一个弹力织物，以确保所有的导管的ð电极母羊的主体。 </li><li>停止全身麻醉，并拔管母羊一次喉反射已恢复正常。 </li><li>返回母羊，以代谢笼有一次她是稳定的全身麻醉。母羊将驻留在代谢笼在实验的持续时间。母羊应能站立，躺下来吃自由采食 ，同时监测非麻醉胎儿无镇静母亲。 </li><li>为以下三个天，施用抗生素预防的母羊（甲氧苄啶磺胺5毫克/千克）和胎儿（250毫克氨苄青霉素经由羊膜导管静脉内，并再次）。 </li><li>评估这两个母羊和胎儿用血气分析的代谢状态。 </li><li>同花顺肝素盐水可能的最小体积的所有导管。注意 - 不要超过每日剂量肝素和流体允许对胎儿。是可能的流体超负荷胎儿。慢慢地冲洗一次每天〜5毫升氯化钠每行预防性应用抗生素后。 </li></ol></li><li>数据记录和分析<ol><li>在手术过程中，任选地记录母亲和胎儿心电图和心脏速率以及母体动脉血压和通气（爪）连续地（ 图1）。用一生窗口监视器来获得心电图除外孕产妇的数据。喂这些数据转换成模拟 - 数字转换器以及胎儿和母体ECG信号;先通过母体和胎儿心电到1901前置放大器。记录和显示制造商的软件中的所有数据。 </li><li> （插入第一动脉导管后立即），并关闭所述子宫后从母羊和胎儿为动脉血气分析，乳酸盐，葡萄糖同时取1毫升动脉样品和碱剩余测定（以等离子体）在胎儿手术的开始。 </li><li>期间术后恢复，取3毫升胎儿血液样品来测量IL-6和TNF-α的inflammatory配置文件。离心机在4℃下的等离子体（4分钟，4000×g离心），滗和血浆储存在-80℃用于后续的ELISA检测。 注意：对于所报告的代表性结果的目的，手术后六天动物使用静脉注射的20毫升的戊巴比妥钠处死。胎儿的生长是由身体，大脑，肝脏和产妇的权重评估。实验期间的持续时间将明显取决于选择用于特定研究问题的设计，并且可以达到〜6周。 </li></ol></li><li>细胞因子分析（可选步骤） <ol><li>确定使用绵羊特异性的夹心ELISA的细胞因子浓度（IL-6，TNF-α）在血浆中。预涂层的小鼠抗羊的单克隆抗体（捕获抗体的IL-6）或小鼠抗牛单克隆抗体（TNF-α）的浓度上ELISA板4微克/毫升，在4℃下O / N，经过3次洗涤用洗涤缓冲液（0.05％吐温20的PBS，PBST）。 </li><li>阻挡板与在PBST中的1％BSA 1小时。用洗涤缓冲液洗涤板3次。 </li><li>使用重组羊蛋白（IL-6，TNF-α），为的ELISA标准，制备连续稀释的范围从标准的1 2000的1ng / ml至7 31.25标准皮克/毫升。 </li><li>负载50微升串行稀释蛋白质标准和每孔样品，孵育2小时，在室温，洗净板3次。运行所有标准和样品一式两份。 </li><li>申请50微升的兔抗绵羊多克隆抗体（检测抗体，IL-6）或兔抗牛多克隆抗体（TNF-α）的稀释度为1：在井250和孵育在室温30分钟。用洗涤缓冲液洗平板5次。 </li><li>加入50微升的山羊抗兔IgG-HRP缀合的（稀释1：5000）的30分钟。 </li><li>孵育用50μl每孔的TMB底物溶液。 </li><li>停止显色反应物在所希望的时间，用25微升2N硫酸。 </li><li>阅读PL茨上ELISA板读数器在450nm处，用一个570纳米波长校正。 注意：在我们的测定中，IL-6的ELISA的灵敏度为16微克/毫升，灵敏度的TNF-α酶联免疫吸附率为13.9皮克/毫升。对于所有的测定，方差的批内和批间的系数为&lt;5％和&lt;10％之间。 </li></ol></li><li>统计分析 注意：确切的统计方法将取决于研究的问题。这里，我们报告用于测试在报告在表2中的血液气体任何显著差异的方法。 <ol><li>测试使用Kolmogorov-Smirnov检验，随后的参数或非参数检验与校正多重比较正常数据分布，根据。 </li><li>使用K-均值聚类分析，以确定那名自发缺氧的人群的胎儿，并确定相应的PO 2和O 2饱和值（考虑PO 2 &lt;20 mmHg或O 2星期六&lt;55％为羊升缺氧）44-48 </li><li>使用统计软件，如SPSS的分析。 </li><li>目前的数据为平均±SD用在P &lt;0.05认为显著统计学差异。 </li></ol></li></ol>

No disclosures have been made.

麻醉及手术过程中呈现所需要的建立动物模型，为研究胎儿生理和病理生理：长期仪器非麻醉的胎羊。 该协议中的四个关键步骤应该强调。首先，通过母亲侧面传递导管和电极：重要的是，这是在一次以避免任何内部器官损伤。第二，固定之前外缝合胎儿uterotomy运行地点：这是至关重要的，以防止或尽量减少羊水和前子宫闭合羊膜随后缝合的损失。三，动脉插管：胎羊动脉?...

对正常和损害怀孕研究各种动物模型的存在，包括实验室啮齿类，非人类的灵长类动物和家养反刍动物。1,2,3,4,5长期仪器妊娠母羊已被广泛使用50年人类胎儿发育和答复的模型的病理生理刺激如脂多糖（LPS）。6-10以下的LPS暴露病变模仿正是由于这两个物种的一个类似的成熟信息见于早产儿脑室周围白质软化，这是。 11，12 其他妊娠并发症也已经研究的很详细，如发现产前糖皮质激素促进肺的发育13-15和了解胎儿宫内发育迟缓（IUGR）对胎儿16,17的影响。 广泛使用的胎羊模型的是由于uniq的非麻醉胎羊到外科位置和维护导管和电极的UE顺从，允许重复的血液采样，记录的生物电活动的，应用电刺激和体内脑成像。18遥测也是可能的，虽然不经常使用的但由于较高的复杂性来设置，以及初始和维护成本。19 此外，胎羊模型是非常通用的仪器的许多变化是可能根据目标的措施。例如，有可能在数天来记录到周实时多元信号如胎儿呼吸运动，电脑活动，心血管反应，心电图，使用流量探针或微球体等由于局部血流的范围器官这种多功能性，已经进行了包括CARDI的发展广泛的研究ovascular系统20,21，下丘脑-垂体-肾上腺轴（HPA）22，大脑发育23和睡眠状态的发展，特别是24，缺氧的影响/窒息25，低温治疗26，炎症6-11，无论是27的组合，糖皮质激素28,29，抗抑郁药30，支气管肺发育不良（BPD）31,32，胎儿编程33,34,35,36,37,38,39或新颖的胎儿监护模式开发之前和分娩期间的名字，但调查的几个领域。40,41,42,43 提出的方法的总体目标是展示这种多功能车型的基本实现。其允许建立多种研究胎儿生理和病理生理上一体化，器官，细胞和分子水平的急性和慢性实验方案。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>ACE Light source</td>
			<td>Schott-Fostec</td>
			<td>A20500</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting scissors</td>
			<td>Fine Science Tools</td>
			<td>14060 - 11</td>
			<td></td>
		</tr>
		<tr>
			<td>Angled dissecting scissors</td>
			<td>Fine Science Tools</td>
			<td>15006 - 09</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel handle</td>
			<td>Fine Science Tools</td>
			<td>10003 - 12</td>
			<td>alternating dissecting tool</td>
		</tr>
		<tr>
			<td>Curved scalpel blades #12</td>
			<td>Fine Science Tools</td>
			<td>10012 - 00</td>
			<td>alternating dissecting tool</td>
		</tr>
		<tr>
			<td>Bone scissors</td>
			<td>Fine Science Tools</td>
			<td>16044 - 10</td>
			<td></td>
		</tr>
		<tr>
			<td>S &#38; T suture tying forceps</td>
			<td>Fine Science Tools</td>
			<td>00272 - 13</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont SS forceps - angled</td>
			<td>Fine Science Tools</td>
			<td>11203 - 25&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Braided silk suture size 6-0</td>
			<td>Teleflex Medical</td>
			<td>07 - 30&#160; - 10</td>
			<td></td>
		</tr>
		<tr>
			<td>Medical Tape</td>
			<td>transpore</td>
			<td>3M</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine hydrochloride 100 mg/ml</td>
			<td>Hospira</td>
			<td>NDC 0409 - 2051 - 05</td>
			<td>Final Does is 80 mg/kg</td>
		</tr>
		<tr>
			<td>Tranqui Ved Injection (xylazine 100 mg/ml)</td>
			<td>Vecdo</td>
			<td>NDC 50989 - 234 - 11</td>
			<td>Final Does is 10 mg/kg</td>
		</tr>
		<tr>
			<td>Reactive orange 14</td>
			<td>Sigma - Aldrich</td>
			<td>R - 8254</td>
			<td></td>
		</tr>
		<tr>
			<td>Ringers Solution Components</td>
			<td></td>
			<td></td>
			<td>Solution is gas equilibrated with 95% O2 and 5% Co2, final pH 7.4</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma - Aldrich</td>
			<td>S7653</td>
			<td>Final Concentration: 118 mM</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Fisher Scientific</td>
			<td>P217 - 3</td>
			<td>Final Concentration: 4.7 mM</td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate</td>
			<td>Fisher Scientific</td>
			<td>C79 - 500</td>
			<td>Final Concentration: 2.5 mM</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Fisher Scientific</td>
			<td>P -285</td>
			<td>Final Concentration: 1.2 mM</td>
		</tr>
		<tr>
			<td>Magnesium sulfate</td>
			<td>J.T. Baker</td>
			<td>Jan-00</td>
			<td>Final Concentration: 0.57 mM</td>
		</tr>
		<tr>
			<td>4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES)</td>
			<td>Fisher Scientific</td>
			<td>BP 310 - 500</td>
			<td>Final Concentration: 5.95 g/L</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma - Aldrich</td>
			<td>G8270</td>
			<td>Final Concentration: 5.5 mM</td>
		</tr>
		<tr>
			<td>LifeWindow</td>
			<td>Digicare Biomedical Technology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CED bioamplifier and ADC units</td>
			<td>Cambridge Electronic Design Limited, 
			Unit 4, Science Park, 
			Milton Road, 
			Cambridge CB4 0FE 
			ENGLAND.</td>
			<td>Bioamp: 1902; ADC: micro1401; Data acquisition software: Spike 2, V7.13</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurolog analog signal bioamplifier</td>
			<td>Digitimer Ltd 
			37 Hydeway 
			Welwyn Garden City 
			Hertfordshire, AL7 3BE, England</td>
			<td>NL108A</td>
			<td></td>
		</tr>
		<tr>
			<td>ABL800Flex</td>
			<td>Radiometer Canada; 200 Aberdeen Dr, London, ON N5V 4N2</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf 5804R</td>
			<td>Eppendorf Canada; 2810 Argentia Road, #2 
			Mississauga, Ontario, L5N 8L2</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Arrow Jugular Catheterization Set</td>
			<td>Arrow International, Inc., 2400 Bernville Road, Reading, PA 19605 USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Atravet</td>
			<td></td>
			<td></td>
			<td>10 mg/mL</td>
		</tr>
		<tr>
			<td>Diazepam</td>
			<td></td>
			<td></td>
			<td>5mg/mL</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Ketalar</td>
			<td></td>
			<td>100 mg/mL</td>
		</tr>
		<tr>
			<td>Propofol</td>
			<td></td>
			<td></td>
			<td>10 mg/mL</td>
		</tr>
		<tr>
			<td>SurgiVeT</td>
			<td>Endotracheal Tubes; Smiths Medical ASD, Inc. St. Paul, MN 55112, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cook Airway Exchange Catheter with RAPI-FIT Adapters</td>
			<td>Cook Critical Care 750, Bloomington IN 47402-0489 USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dispomed Ventilator</td>
			<td>Dispomed Ltd., 745 Nazaire-Laurin, Joliette, Quebec J6E 0L6</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD Insyte-W</td>
			<td>Becton Dickinson, Infusion Therapy Systems Inc., 9450 S State St, Sandy Utah 84070 USA</td>
			<td></td>
			<td>22 to 20 G; 1 in [0.9 x 25 mm] to 1.16 in [1.1 x 30 mm]</td>
		</tr>
		<tr>
			<td>Edwards Lifesciences Ref: PX272 Pressure monitoring kit with TruWave Disposable Pressure</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LifeWindow LW6000</td>
			<td>Digicare Biomedical Technology 107 Commerce Road, Boynton Beach, FL 33426-9365 USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gaymar</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Babcock</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Polyvinyl catheters</td>
			<td>SCI (Scientific Commodities Inc.)</td>
			<td></td>
			<td>2 meters</td>
		</tr>
		<tr>
			<td>2-0 Vicryl</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Castroviejo scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>electrocardiogram (ECG)</td>
			<td>LIFYY, Metrofunk Kabel-Union, Berlin, Germany</td>
			<td></td>
			<td>four copper electrodes in single sheath, 2 meters</td>
		</tr>
		<tr>
			<td>2-O Vicryl</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3-0 Vicryl</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PDS II USP</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trimethoprim sulfadoxine</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stopcock</td>
			<td>Argon Medical, Cat&#160;041220001A</td>
			<td></td>
			<td>Double 4-way Stopcock with male luer lock</td>
		</tr>
		<tr>
			<td>Needles</td>
			<td>Tyco Healthcare 8881202389</td>
			<td></td>
			<td>Monoject aluminum hub blunt needles, 22Gx, 0.7mmx 38.1mm: for fetal arterial and venous catheters</td>
		</tr>
		<tr>
			<td>Needles</td>
			<td>Tyco Healthcare 8881202322</td>
			<td></td>
			<td>Monoject aluminum hub blunt needles, 16Gx, 1.6mmx38.1mm: for amniotic catheters</td>
		</tr>
	</tbody>
</table>

instrumentation of near-term fetal sheep for multivariate chronic non-anesthetized recordings

The chronically instrumented pregnant sheep has been used as a model of human fetal development and responses to pathophysiologic stimuli such as endotoxins, bacteria, umbilical cord occlusions, hypoxia and various pharmacological treatments. The life-saving clinical practices of glucocorticoid treatment in fetuses at risk of premature birth and the therapeutic hypothermia have been developed in this model. This is due to the unique amenability of the non-anesthetized fetal sheep to the surgical placement and maintenance of catheters and electrodes, allowing repetitive blood sampling, substance injection, recording of bioelectrical activity, application of electric stimulation and in vivo organ imaging. Here we describe the surgical instrumentation procedure required to achieve a stable chronically instrumented non-anesthetized fetal sheep model including characterization of the post-operative recovery from blood gas, metabolic and inflammation standpoints.

38怀孕的时间，日期母羊仪器在128±2天的妊娠期（DGA，〜0.88妊娠，期限145 DGA）与动脉，静脉和羊膜导管和心电图（ECG）与全身麻醉下无菌技术（包括母羊和胎儿电极）。如果双胎妊娠的胎儿较大基于触诊和估计跨期直径选择;替代地，胎儿必须被启动，可以随机选择，以避免任何可能的偏差或两者胎儿可能进行检测。该过程的总持续时间为124±27分钟。胎儿上半身必须被启动的部分仍然是子宫外为...

Watch this Scientific Journal Video about Instrumentation of Near-term Fetal Sheep for Multivariate Chronic Non-anesthetized Recordings at JoVE.com

Instrumentation of Near-term Fetal Sheep for Multivariate Chronic Non-anesthetized Recordings

慢性病仪表非麻醉的胎羊模型被用于研究在健康和疾病的人类胎儿发育，因为它允许外科安置和维护导管和电极，反复血液采样，物质喷射，记录的生物电活动的，以及在体内成像 。我们描述了建立这种模式所需的程序。

仪表近期胎羊多元慢性非麻醉的录音

The chronically instrumented pregnant sheep has been used as a model of human fetal development and responses to pathophysiologic stimuli such as ...

instrumentation-near-term-fetal-sheep-for-multivariate-chronic-non

Research

JoVE Journal

Developmental Biology

9.4K 次观看.  CHUV, Université de Montréal, St-Hyacinthe, QC. 慢性病仪表非麻醉的胎羊模型被用于研究在健康和疾病的人类胎儿发育，因为它允许外科安置和维护导管和电极，反复血液采样，物质喷射，记录的生物电活动的，以及在体内成像 。我们描述了建立这种模式所需的程序。 

视频: Instrumentation of Near-term Fetal Sheep for Multivariate Chronic Non-anesthetized Recordings

Preparation and Culture of Myogenic Precursor Cells/Primary Myoblasts from Skeletal Muscle of Adult and Aged Humans

Skeletal muscle homeostasis depends on muscle growth (hypertrophy), atrophy and regeneration. During ageing and in several diseases, muscle wasting occurs. Loss of muscle mass and function is associated with muscle fiber type atrophy, fiber type switching, defective muscle regeneration associated with dysfunction of satellite cells, muscle stem cells, and other pathophysiological processes. These changes are associated with changes in intracellular as well as local and systemic niches. In addition to most commonly used rodent models of muscle ageing, there is a need to study muscle homeostasis and wasting using human models, which due to ethical implications, consist predominantly of in vitro cultures. Despite the wide use of human Myogenic Progenitor Cells (MPCs) and primary myoblasts in myogenesis, there is limited data on using human primary myoblast and myotube cultures to study molecular mechanisms regulating different aspects of age-associated muscle wasting, aiding in the validation of mechanisms of ageing proposed in rodent muscle. The use of human MPCs, primary myoblasts and myotubes isolated from adult and aged people, provides a physiologically relevant model of molecular mechanisms of processes associated with muscle growth, atrophy and regeneration. Here we describe in detail a robust, inexpensive, reproducible and efficient protocol for the isolation and maintenance of human MPCs&#160;and their progeny&#160;&#8212; myoblasts and myotubes from human muscle samples using enzymatic digestion. Furthermore, we have determined the passage number at which primary myoblasts from adult and aged people undergo senescence in an in vitro culture. Finally, we show the ability to transfect these myoblasts and the ability to characterize their proliferative and differentiation capacity and propose their suitability for performing functional studies of molecular mechanisms of myogenesis and muscle wasting in vitro.

This protocol describes a robust, reproducible and simple method of isolation and culture of myoblast progenitor cells from the skeletal muscle of adult and aged people. The muscles used here include foot and leg muscles. This approach enables the isolation of an enriched population of primary myoblasts for functional studies.

制备及文化活动从成年和老年人类骨骼肌成肌前体细胞/成肌细胞主要的

Skeletal muscle homeostasis depends on muscle growth (hypertrophy), atrophy and regeneration. During ageing and in several diseases, muscle wasting ...

科研

发育生物学

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly gained attention because they do not pose any ethical or moral concerns. Current methods to isolate MSCs from UC yield low amounts of cells with variable proliferation potentials. Since UC is an anatomically-complex organ, differences in MSC properties may be due to the differences in the anatomical regions of their isolation. In this study, we first dissected the cord/placenta samples into three discrete anatomical regions: UC, cord-placenta junction (CPJ), and fetal placenta (FP). Second, two distinct zones, cord lining (CL) and Wharton&#39;s jelly (WJ), were separated. The explant culture technique was then used to isolate cells from the four sources. The time required for the primary culture of cells from the explants varied depending on the source of the tissue. Outgrowth of the cells occurred within 3 - 4 days of the CPJ explants, whereas growth was observed after 7 - 10 days and 11 - 14 days from CL/WJ and FP explants, respectively. The isolated cells were adherent to plastic and displayed fibroblastoid morphology and surface markers, such as CD29, CD44, CD73, CD90, and CD105, similarly to bone marrow (BM)-derived MSCs. However, the colony-forming efficiency of the cells varied, with CPJ-MSCs and WJ-MSCs showing higher efficiency than BM-MSCs. MSCs from all four sources differentiated into adipogenic, chondrogenic, and osteogenic lineages, indicating that they were multipotent. CPJ-MSCs differentiated more efficiently in comparison to other MSC sources. These results suggest that the CPJ is the most potent anatomical region and yields a higher number of cells, with greater proliferation and self-renewal capacities in vitro. In conclusion, the comparative analysis of the MSCs from the four sources indicated that CPJ is a more promising source of MSCs for cell therapy, regenerative medicine, and tissue engineering.

Here, we present a protocol for the dissection of human umbilical cord (UC) and fetal placenta sample into cord lining (CL), Wharton&#39;s jelly (WJ), cord-placenta junction (CPJ), and fetal placenta (FP) for the isolation and characterization of mesenchymal stromal cells (MSCs) using the explant culture technique.

从人类脐带和胎盘胎儿分离和鉴定间充质干细胞

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

Whole-cell Patch-clamp Recordings of Isolated Primary Epithelial Cells from the Epididymis

The epididymis is an essential organ for sperm maturation and reproductive health. The epididymal epithelium consists of intricately connected cell types that are distinct not only in molecular and morphological features but also in physiological properties. These differences reflect their diverse functions, which together establish the necessary microenvironment for the post-testicular sperm development in the epididymal lumen. The understanding of the biophysical properties of the epididymal epithelial cells is critical for revealing their functions in sperm and reproductive health, under both physiological and pathophysiological conditions. While their functional properties have yet to be fully elucidated, the epididymal epithelial cells can be studied using the patch-clamp technique, a tool for measuring the cellular events and the membrane properties of single cells. Here, we describe the methods of cell isolation and whole-cell patch-clamp recording to measure the electrical properties of primary dissociated epithelial cells from the rat cauda epididymides.

We present a protocol that combines cell isolation and whole-cell patch-clamp recording to measure the electrical properties of the primary dissociated epithelial cells from the rat cauda epididymides. This protocol allows for investigation of the functional properties of primary epididymal epithelial cells to further elucidate the physiological role of the epididymis.

分离的原发性上皮细胞从附睾的全细胞贴片钳记录

The epididymis is an essential organ for sperm maturation and reproductive health. The epididymal epithelium consists of intricately connected cell types ...

Zika Virus Infection of Cultured Human Fetal Brain Neural Stem Cells for Immunocytochemical Analysis

Human fetal brain neural stem cells are a unique non-genetically modified model system to study the impact of various stimuli on human developmental neurobiology. Rather than use an animal model or genetically modified induced pluripotent cells, human neural stem cells provide an effective in vitro system to examine the effects of treatments, screen drugs, or examine individual differences. Here, we provide the detailed protocols for methods used to expand human fetal brain neural stem cells in culture with serum-free media, to differentiate them into various neuronal subtypes and astrocytes via different priming procedures, and to freeze and recover these cells. Furthermore, we describe a procedure of using human fetal brain neural stem cells to study Zika virus infection.

This article details the methods that are used to expand human fetal brain neural stem cells in culture, as well as how to differentiate them into various neuronal subtypes and astrocytes, with an emphasis on the use of neural stem cells to study Zika virus infection.

人胎脑神经干细胞 Zika 病毒感染的免疫细胞化学分析

Human fetal brain neural stem cells are a unique non-genetically modified model system to study the impact of various stimuli on human developmental ...

Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model

In utero transplantation (IUT) is a unique and versatile mode of therapy that can be used to introduce stem cells, viral vectors, or any other substances early in the gestation. The rationale behind IUT for therapeutic purposes is based on the small size of the fetus, the fetal immunologic immaturity, the accessibility and proliferative nature of the fetal stem or progenitor cells, and the potential to treat a disease or the onset of symptoms prior to birth. Taking advantage of these normal developmental properties of the fetus, the delivery of hematopoietic stem cells (HSC) via an IUT has the potential to treat congenital hematologic disorders such as sickle cell disease, without the required myeloablative or immunosuppressive conditioning required for postnatal HSC transplants. Similarly, the accessibility of progenitor cells in multiple organs during development potentially allows for a more efficient targeting of stem/progenitor cells following an IUT of viral vectors for gene therapy or genome editing. Additionally, IUT can be used to study normal developmental processes including, but not limited to, the development of immunologic tolerance. The murine model provides a valuable and affordable means to understanding the potential and limitations of IUT prior to pre-clinical large animal studies and an eventual clinical application. Here, we describe a protocol for performing an IUT in the murine fetus through intravenous and intra-amniotic routes. This protocol has been used successfully to elucidate the necessary conditions and mechanisms behind in utero hematopoietic stem cell transplantation, tolerance induction, and in utero gene therapy.

Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model

We describe a protocol for performing an in utero transplantation (IUT) through intravenous and intra-amniotic routes of injection in the murine model. This protocol can be used to introduce cells, viral vectors, and other substances into the unique immune-tolerant fetal environment.

小鼠宫内移植中静脉和羊膜内植入的研究

In utero transplantation (IUT) is a unique and versatile mode of therapy that can be used to introduce stem cells, viral vectors, or any other substances ...

High Frequency Ultrasound for the Analysis of Fetal and Placental Development In Vivo

Ultrasound imaging is a widespread method used to detect organ anomalies and tumors in human and animal tissues. The method is non-invasive, harmless, and painless, and the application is easy, fast, and can be done anywhere, even with mobile devices. During pregnancy, ultrasound imaging is standardly used to closely monitor fetal development. The technique is important to assess intrauterine growth restriction (IUGR), a pregnancy complication with short- and long-term health consequences for both the mother and fetus. Understanding the process of IUGR is indispensable for developing effective therapeutic strategies. 
The ultrasound system used in this manuscript is an ultrasound device produced for the analysis of small animals and can be used in various research fields, including pregnancy research. Here we describe the usage of the system for in vivo analysis of fetuses from natural killer (NK) cell/mast cell (MC)-deficient mothers that give birth to growth-restricted pups. The protocol includes preparation of the system, handling of the mice before and during measurements, and the usage of the B-mode, color doppler mode, and pulse-wave doppler mode. Fetal size, placental size, and blood supply to the fetus were analyzed. We found reduced implantation sizes and smaller placentas in NK/MC-deficient mice from mid-gestation onwards. In addition, MC/NK-deficiency was associated with absent and reversed end diastolic flow in the fetal Arteria umbilicalis(UmA) and an elevated resistance index. The methods described in the protocol can easily be used for related and non-related research topics.

High Frequency Ultrasound for the Analysis of Fetal and Placental Development In Vivo

Here we describe the technique of high frequency ultrasound for in vivo analysis of fetuses in mice. This method allows the follow-up of fetuses and the analysis of placental parameters as well as maternal and fetal blood flow throughout pregnancy.

高频超声在胎儿和胎盘发育分析中的影响

Ultrasound imaging is a widespread method used to detect organ anomalies and tumors in human and animal tissues. The method is non-invasive, harmless, and ...

Culturing and Measuring Fetal and Newborn Murine Long Bones

Long bones are complex and dynamic structures, which arise from endochondral ossification via a cartilage intermediate. The limited access to healthy human bones makes particularly valuable the use of mammalian models, such as mouse and rat, to look into different aspects of bone growth and homeostasis. Additionally, the development of sophisticated genetic tools in mice allows more complex studies of long bone growth and asks for an expansion of techniques used to study bone growth. Here, we present a detailed protocol for ex vivo murine bone culture, which allows the study of bone and cartilage in a tightly controlled manner while recapitulating most of the in vivo process. The method described allows the culture of a range of bones, including tibia, femur, and metatarsal bones, but we have focused mainly on tibial culture here. Moreover, it can be used in combination with other techniques, such as time-lapse live imaging or drug treatment.

Here, we present a method for ex vivo culture of long murine bones at both fetal and newborn stages, suitable for analyzing bone and cartilage development and homeostasis in controlled conditions while recapitulating the in vivo process.

胎儿和新生儿小鼠长骨的培养与测量

Long bones are complex and dynamic structures, which arise from endochondral ossification via a cartilage intermediate. The limited access to healthy ...

Recapitulating Suckling-to-Weaning Transition In Vitro using Fetal Intestinal Organoids

At the end of the suckling period, many mammalian species undergo major changes in the intestinal epithelium that are associated with the capability to digest solid food. This process is termed suckling-to-weaning transition and results in the replacement of neonatal epithelium with adult epithelium which goes hand in hand with metabolic and morphological adjustments. These complex developmental changes are the result of a genetic program that is intrinsic to the intestinal epithelial cells but can, to some extent, be modulated by extrinsic factors. Prolonged culture of mouse primary intestinal epithelial cells from late fetal period, recapitulates suckling-to-weaning transition in vitro. Here, we describe a detailed protocol for mouse fetal intestinal organoid culture best suited to model this process in vitro. We describe several useful assays designed to monitor the change of intestinal functions associated with suckling-to-weaning transition over time. Additionally, we include an example of an extrinsic factor that is capable to affect suckling-to-weaning transition in vivo, as a representation of modulating the timing of suckling-to-weaning transition in vitro. This in vitro approach can be used to study molecular mechanisms of the suckling-to-weaning transition as well as modulators of this process. Importantly, with respect to animal ethics in research, replacing in vivo models by this in vitro model contributes to refinement of animal experiments and possibly to a reduction in the use of animals to study gut maturation processes.

This protocol describes how to mimic suckling-to-weaning transition in vitro using mouse late fetal intestinal organoids cultured for 30 days.

使用胎儿肠道有机体在体外重述吸吮到发胶过渡

At the end of the suckling period, many mammalian species undergo major changes in the intestinal epithelium that are associated with the capability to ...

Transuterine Fetal Tracheal Occlusion Model in Mice

Fetal tracheal occlusion (TO), an established treatment modality, promotes fetal lung growth and survival in severe congenital diaphragmatic hernia (CDH). Following TO, retention of the secreted epithelial fluid increases luminal pressure and induces lung growth. Various animal models have been defined to understand the pathophysiology of CDH and TO. All have their own advantages and disadvantages such as the difficulty of the technique, the size of the animal, cost, high mortality rates, and the availability of genetic tools. Herein, a novel transuterine model of murine fetal TO is described. Pregnant mice were anesthetized, and the uterus exposed via a midline laparotomy. The trachea of selected fetuses were ligated with a single transuterine suture placed behind the trachea, one carotid artery, and one jugular vein. The dam was closed and allowed to recover. Fetuses were collected just before parturition. Lung to body weight ratio in TO fetuses was higher than that in control fetuses. This model provides researchers with a new tool to study the impact of both TO and increased luminal pressure on lung development.

Various animal models of congenital diaphragmatic hernia and fetal tracheal occlusion present advantages and disadvantages regarding ethical issues, cost, surgical difficulty, size, survival rates, and availability of genetic tools. This model provides a new tool to study the impact of both tracheal occlusion and increased luminal pressure on lung development.

小鼠的转宫胎儿气管闭塞模型

Fetal tracheal occlusion (TO), an established treatment modality, promotes fetal lung growth and survival in severe congenital diaphragmatic hernia (CDH). ...

Vitrification of In Vitro Matured Oocytes Collected from Adult and Prepubertal Ovaries in Sheep

In livestock, in vitro embryo production systems can be developed and sustained thanks to the large number of ovaries and oocytes that can be easily obtained from a slaughterhouse. Adult ovaries always bear several antral follicles, while in pre-pubertal donors the maximal numbers of oocytes are available at 4 weeks of age, when ovaries bear peak numbers of antral follicles. Thus, 4 weeks old lambs are considered good donors, even if the developmental competence of prepubertal oocytes is lower compared to their adult counterpart. 
Basic research and commercial applications would be boosted by the possibility of successfully cryopreserving vitrified oocytes obtained from both adult and prepubertal donors. The vitrification of oocyte collected from prepubertal donors would also allow shortening the generation interval and thus increasing the genetic gain in breeding programs. However, the loss of developmental potential after cryopreservation makes mammalian oocytes probably one of the most difficult cell types to cryopreserve. Among the available cryopreservation techniques, vitrification is widely applied to animal and human oocytes. Despite recent advancements in the technique, exposures to high concentrations of cryoprotective agents as well as chilling injury and osmotic stress still induce several structural and molecular alterations and reduce the developmental potential of mammalian oocytes. Here, we describe a protocol for the vitrification of sheep oocytes collected from juvenile and adult donors and matured in vitro prior to cryopreservation. The protocol includes all the procedures from oocyte in vitro maturation to vitrification, warming and post-warming incubation period. Oocytes vitrified at the MII stage can indeed be fertilized following warming, but they need extra time prior to fertilization to restore damage due to cryopreservation procedures and to increase their developmental potential. Thus, post-warming culture conditions and timing are crucial steps for the restoration of oocyte developmental potential, especially when oocyte are collected from juvenile donors.

The protocol aims at providing a standard method for the vitrification of adult and juvenile sheep oocytes. It includes all the steps from the preparation of the in vitro maturation media to the post-warming culture. Oocytes are vitrified at the MII stage using Cryotop to ensure the minimum essential volume.

从绵羊的成人和前卵巢收集的体外成熟卵母细胞的振动

In livestock, in vitro embryo production systems can be developed and sustained thanks to the large number of ovaries and oocytes that can be easily ...