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>

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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:胎羊动脉是约1-2?...

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><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 &amp;amp; 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&amp;nbsp;</td><td></td></tr><tr><td>Braided silk suture size 6-0</td><td>Teleflex Medical</td><td>07 - 30&amp;nbsp; - 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,&lt;br/&gt; Unit 4, Science Park,&lt;br/&gt; Milton Road,&lt;br/&gt; Cambridge CB4 0FE&lt;br/&gt; 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&lt;br/&gt; 37 Hydeway&lt;br/&gt; Welwyn Garden City&lt;br/&gt; 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&lt;br/&gt; 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>5 mg/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&amp;nbsp;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

Three-Dimensional Ultrasonic Needle Tip Tracking with a Fiber-Optic Ultrasound Receiver

Ultrasound is frequently used for guiding minimally invasive procedures, but visualizing medical devices is often challenging with this imaging modality. When visualization is lost, the medical device can cause trauma to critical tissue structures. Here, a method to track the needle tip during ultrasound image-guided procedures is presented. This method involves the use of a fiber-optic ultrasound receiver that is affixed within the cannula of a medical needle to communicate ultrasonically with the external ultrasound probe. This custom probe comprises a central transducer element array and side element arrays. In addition to conventional two-dimensional (2D) B-mode ultrasound imaging provided by the central array, three-dimensional (3D) needle tip tracking is provided by the side arrays. For B-mode ultrasound imaging, a standard transmit-receive sequence with electronic beamforming is performed. For ultrasonic tracking, Golay-coded ultrasound transmissions from the 4 side arrays are received by the hydrophone sensor, and subsequently the received signals are decoded to identify the needle tip&#39;s spatial location with respect to the ultrasound imaging probe. As a preliminary validation of this method, insertions of the needle/hydrophone pair were performed in clinically realistic contexts. This novel ultrasound imaging/tracking method is compatible with current clinical workflow, and it provides reliable device tracking during in-plane and out-of-plane needle insertions.

Accurate and efficient visualization of invasive medical devices is extremely important in many ultrasound-guided minimally invasive procedures. Here, a method for localizing the spatial position of a needle tip relative to the ultrasound imaging probe is presented.

基于光纤超声接收机的三维超声针尖跟踪

Ultrasound is frequently used for guiding minimally invasive procedures, but visualizing medical devices is often challenging with this imaging modality. ...

科研

工程学

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease and injury regional estimates, World Health Organization, 2004). Surgeries that often compromise the quality of life are required to correct heart defects, reminding us of the importance of finding the causes of CHD. Mutant mouse models and live imaging technology have become essential tools to study the etiology of this disease. Although advanced methods allow live imaging of abnormal hearts in embryos, the physiological and hemodynamic states of the latter are often compromised due to surgical and/or lengthy procedures. Noninvasive ultrasound imaging, however, can be used without surgically exposing the embryos, thereby maintaining their physiology. Herein, we use simple M-mode ultrasound to assess heart rates of embryos at E18.5 in utero. The detection of abnormal heart rates is indeed a good indicator of dysfunction of the heart and thus constitutes a first step in the identification of developmental defects that may lead to heart failure.

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Ultra-high frequency ultrasound is a powerful live imaging tool to examine cardiac abnormalities in small animals. Its noninvasiveness allows maintaining the physiologic state of embryos. Herein, we show the use of M-mode ultrasound to measure the heart rates of embryos at E18.5 in utero.

心率在小鼠中通过无创M型超声心动图测量子宫内

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease ...

医学

In Vivo Real-Time Study of Drug Effects on Carotid Blood Flow in the Ovine Fetus

The ability of an organism to maintain a constant blood flow to the brain in response to sudden surges in systemic blood pressure (BP) is known as cerebral autoregulation (CAR), which occurs in the carotid artery. In contrast to full-term neonates, preterm neonates are unable to reduce the cerebral blood flow (CBF) in response to increased systemic BP. In preterm neonates, this exposes the fragile cerebral vessels to high perfusion pressures, leading to their rupture and brain damage. Ex vivo studies using wire myography have demonstrated that carotid arteries from near-term fetuses constrict in response to the activation of adrenergic alpha1 receptors. This response is blunted in the preterm fetus. Thus, to examine the role of alpha1-AR in vivo, presented here is an innovative approach to determine the effects of drugs on a carotid arterial segment in vivo in an ovine fetus during the developmental progression of gestation. The presented data demonstrate the simultaneous measurement of fetal blood flow and blood pressure. The perivascular delivery system can be used to conduct a long-term study over several days. Additional applications for this method could include viral delivery systems to alter the expression of genes in a segment of the carotid artery. These methods could be applied to other blood vessels in the growing organism in utero as well as in adult organisms.

In Vivo Real-Time Study of Drug Effects on Carotid Blood Flow in the Ovine Fetus

The present protocol describes a method to deliver drugs and gene expression-modifying agents perivascularly in an in utero developing fetus. Importantly, the effect of drugs/agents on blood flow can be measured with the progression of pregnancy.

体内 药物对产卵胎颈动脉血流影响的实时研究

The ability of an organism to maintain a constant blood flow to the brain in response to sudden surges in systemic blood pressure (BP) is known as ...

发育生物学

Adaptation of Microelectrode Array Technology for the Study of Anesthesia-induced Neurotoxicity in the Intact Piglet Brain

Every year, millions of children undergo anesthesia for a multitude of procedures. However, studies in both animals and humans have called into question the safety of anesthesia in children, implicating anesthetics as potentially toxic to the brain in development. To date, no studies have successfully elucidated the mechanism(s) by which anesthesia may be neurotoxic. Animal studies allow investigation of such mechanisms, and neonatal piglets represent an excellent model to study these effects due to their striking developmental similarities to the human brain.
This protocol adapts the use of enzyme-based microelectrode array (MEA) technology as a novel way to study the mechanism(s) of anesthesia-induced neurotoxicity (AIN). MEAs enable real-time monitoring of in vivo neurotransmitter activity and offer exceptional temporal and spatial resolution. It is hypothesized that anesthetic neurotoxicity is caused in part by glutamate dysregulation and MEAs offer a method to measure glutamate. The novel implementation of MEA technology in a piglet model presents a unique opportunity for the study of AIN.

This study explores the novel use of enzyme-based microelectrode array (MEA) technology to monitor in vivo neurotransmitter activity in piglets. The hypothesis was that glutamate dysregulation contributes to the mechanism of anesthetic neurotoxicity. Here, we present a protocol to adapt MEA technology to study the mechanism of anesthesia-induced neurotoxicity.

微电极阵列技术在完整仔猪脑麻醉诱导神经毒性研究中的应用

Every year, millions of children undergo anesthesia for a multitude of procedures. However, studies in both animals and humans have called into question ...

神经科学

The Perinatal Asphyxiated Lamb Model: A Model for Newborn Resuscitation

Birth asphyxia accounts for nearly one million deaths worldwide each year, and is one of the primary causes of early neonatal morbidity and mortality. Many aspects of the current neonatal resuscitation guidelines remain controversial given the difficulties in conducting randomized clinical trials owing to the infrequent and often unpredictable need for extensive resuscitation. Most studies on neonatal resuscitation stem from manikin models that fail to truly reflect physiologic changes or piglet models that have cleared their lung fluid and that have completed the transition from fetal to neonatal circulation. The present protocol provides a detailed step-by-step description on how to create a perinatal asphyxiated fetal lamb model. The proposed model has a transitioning circulation and fluid-filled lungs, which mimics human newborns following delivery, and is, therefore, an excellent animal model to study newborn physiology. An important limitation to lamb experiments is the higher associated cost.

Invasive instrumentation of the fetal lamb provides accurate physiologic measurements of the transitioning circulation in a model that closely mimics the newly born infant.

围产儿窒息羔羊模型: 新生儿复苏模型的建立

Birth asphyxia accounts for nearly one million deaths worldwide each year, and is one of the primary causes of early neonatal morbidity and mortality. ...

Subdural Soft Electrocorticography (ECoG) Array Implantation and Long-Term Cortical Recording in Minipigs

Neurological impairments and diseases can be diagnosed or treated using electrocorticography (ECoG) arrays. In drug-resistant epilepsy, these help delineate the epileptic region to resect. In long-term applications such as brain-computer interfaces, these epicortical electrodes are used to record the movement intention of the brain, to control the robotic limbs of paralyzed patients. However, current stiff electrode grids do not answer the need for high-resolution brain recordings and long-term biointegration. Recently, conformable electrode arrays have been proposed to achieve long-term implant stability with high performance. However, preclinical studies for these new implant technologies are needed to validate their long-term functionality and safety profile for their translation to human patients. In this context, porcine models are routinely employed in developing medical devices due to their large organ sizes and easy animal handling. However, only a few brain applications are described in the literature, mostly due to surgery limitations and integration of the implant system on a living animal.
Here, we report the method for long-term implantation (6 months) and evaluation of soft ECoG arrays in the minipig model. The study first presents the implant system, consisting of a soft microfabricated electrode array integrated with a magnetic resonance imaging (MRI)-compatible polymeric transdermal port that houses instrumentation connectors for electrophysiology recordings. Then, the study describes the surgical procedure, from subdural implantation to animal recovery. We focus on the auditory cortex as an example target area where evoked potentials are induced by acoustic stimulation. We finally describe a data acquisition sequence that includes MRI of the whole brain, implant electrochemical characterization, intraoperative and freely moving electrophysiology, and immunohistochemistry staining of the extracted brains.
This model can be used to investigate the safety and function of novel design of cortical prostheses; mandatory preclinical study to envision translation to human patients.

Subdural Soft Electrocorticography ECoG Array Implantation and Long-Term Cortical Recording in Minipigs

Here, we present a method for the long-term performance and safety assessment of soft subdural electrode arrays in a minipig model, describing surgical method and tools, postoperative magnetic resonance imaging, electrophysiology of the auditory cortex, electrochemical properties of the implant, and postmortem immunochemistry.

硬膜下软皮质电图 （ECoG） 阵列植入和小型猪的长期皮质记录

Neurological impairments and diseases can be diagnosed or treated using electrocorticography (ECoG) arrays. In drug-resistant epilepsy, these help ...

生物工程

Noninvasive Electrocardiography in the Perinatal Mouse

Electrocardiography (ECG) has long been relied upon as an effective and reliable method of assessing cardiovascular (and cardiopulmonary) function in both human and animal models of disease. Individual heart rate, rhythm, and regularity, combined with quantitative parameters collected from ECG, serve to assess the integrity of the cardiac conduction system as well as the integrated physiology of the cardiac cycle. This article provides a comprehensive description of the methods and techniques used to perform a noninvasive ECG on perinatal and neonatal mouse pups as early as the first postnatal day, without requiring the use of anesthetics. This protocol was designed to directly address a need for a standardized and repeatable method for obtaining ECG in newborn mice. From a translational perspective, this protocol proves to be entirely effective for characterization of congenital cardiopulmonary defects generated using transgenic mouse lines, and particularly for analysis of defects causing lethality at or during the first postnatal days. This protocol also aims to directly address a gap in the scientific literature to characterize and provide normative data associated with maturation of the early postnatal cardiac conduction system. This method is not limited to a specific postnatal timepoint, but rather allows for ECG data collection in neonatal mouse pups from birth to postnatal day 10 (P10), a window that is of critical importance for modeling human diseases in vivo, with particular emphasis on congenital heart disease (CHD).

Here, we present a noninvasive electrocardiography (ECG) protocol, optimized for early postnatal mice, that does not require the use of anesthetics.

围产期小鼠中的非侵入性心电图

Electrocardiography (ECG) has long been relied upon as an effective and reliable method of assessing cardiovascular (and cardiopulmonary) function in both ...

Wireless Telemetry Device Implantation in a Fontan Ovine Model for Continuous and Long-Term Hemodynamic Monitoring

While the Fontan procedure drastically improves life expectancy for patients with single ventricle, it is well recognized that the resulting circulation causes significant disease burden long term as a consequence of chronically elevated central venous pressures and decreased cardiac output. Chronic Fontan animal models are a valuable asset to studying the late physiological outcomes associated with this operation and a necessary tool in the evaluation of future devices designed to alleviate Fontan failure. However, previous attempts at the creation of chronic Fontan models have been hindered by poor survival rates. Additionally, effective hemodynamic data collection poses a significant challenge in freely moving animals. To this end, the use of wireless implantable telemetry systems provides a novel solution for real-time and long-term monitoring of cardiovascular data. This protocol describes the methodology for surgical implantation of a wireless telemetry device in a Fontan survival ovine model, facilitating the continuous and ongoing recording of several hemodynamic parameters, including heart rate, arterial blood pressure, and localized pressures in the inferior (IVC) and superior vena cava (SVC). Telemetry devices were implanted with cannulation of either the carotid artery and internal jugular vein or femoral artery and vein, for placement of pressure-sensing catheters in the ascending aorta and SVC or abdominal aorta and IVC, respectively. The use of the wireless telemetry systems enabled close postoperative monitoring following a single-stage Fontan operation, which contributed to improved animal welfare and survival.

This protocol describes the surgical methodology for implanting a large animal wireless telemetry device to enable continuous and long-term collection of hemodynamic data, including heart rate, arterial blood pressure, inferior and superior vena cava pressures, and cardiac rhythm.

在 Fontan 绵羊模型中植入无线遥测设备，用于连续和长期血流动力学监测

While the Fontan procedure drastically improves life expectancy for patients with single ventricle, it is well recognized that the resulting circulation ...

Multi-system Monitoring for Identification of Seizures, Arrhythmias and Apnea in Conscious Restrained Rabbits

Patients with ion channelopathies are at a high risk of developing seizures and fatal cardiac arrhythmias. There is a higher prevalence of heart disease and arrhythmias in people with epilepsy (i.e., epileptic heart.) Additionally, cardiac and autonomic disturbances have been reported surrounding seizures. 1:1,000 epilepsy patients/year die of sudden unexpected death in epilepsy (SUDEP). The mechanisms for SUDEP remain incompletely understood. Electroencephalograms (EEG) and electrocardiograms (ECG) are two techniques routinely used in the clinical setting to detect and study the substrates/triggers for seizures and arrhythmias. While many studies and descriptions of this methodology are in rodents, their cardiac electrical activity differs significantly from humans. This article provides a description of a non-invasive method for recording simultaneous video-EEG-ECG-oximetry-capnography in conscious rabbits. As cardiac electrical function is similar in rabbits and humans, rabbits provide an excellent model of translational diagnostic and therapeutic studies. In addition to outlining the methodology for data acquisition, we discuss the analytical approaches for examining neuro-cardiac electrical function and pathology in rabbits. This includes arrhythmia detection, spectral analysis of EEG and a seizure scale developed for restrained rabbits.

Using simultaneous video-EEG-ECG-oximetry-capnography, we developed a methodology to evaluate the susceptibility of rabbit models to develop provoked arrhythmias and seizures. This novel recording system establishes a platform to test the efficacy and safety of therapeutics and can capture the complex cascade of multi-system events that culminate in sudden death.

意识约束兔子中癫痫发作、心律失常和呼吸暂停的多系统监测

Patients with ion channelopathies are at a high risk of developing seizures and fatal cardiac arrhythmias. There is a higher prevalence of heart disease ...

Continuous Telemetric In Utero Tracheal Pressure Measurements in Fetal Lambs

Normal in utero lung development and growth rely upon the expansion of airspaces and the controlled efflux of lung liquid into the amniotic space. Infants with congenital diaphragmatic hernia (CDH) also have lung hypoplasia due to occupation of the chest cavity by the stomach and bowel and, in the most severe cases, the liver. Balloon tracheal occlusion reduces the severity of lung hypoplasia in fetuses with CDH but increases the risk of premature birth. Understanding the optimal occlusion pressure and duration required to improve lung hypoplasia with tracheal occlusion is essential to improving in utero corrective treatments for CDH. The study reports a new method for continuous measurement of the intratracheal and amniotic pressures in an unoccluded and occluded fetal lamb surgical model of CDH. Time-pregnant Merino ewes underwent two recovery hysterotomies: the first at ~80 days of gestation to create the CDH, and the second at ~101 days of gestation to occlude the fetal trachea and implant an intratracheal and amniotic pressure measurement device. Lambs were delivered at ~142 days, and the pressure measurement device was removed and cleaned. The data were downloaded and filtered using a 6 h window. Transrespiratory pressure was calculated.

<meta charset="utf8"></head><body>胎羔羊子宫内气管压力连续遥测测量

Optimization of occlusion pressure and duration required to improve lung hypoplasia with tracheal occlusion is essential to improve in utero corrective treatments for congenital diaphragmatic hernia (CDH). This study reports a new method for continuous measurement of the tracheal pressure in an occluded and unoccluded fetal lamb surgical model of CDH.

胎羔羊子宫内气管压力连续遥测测量

Normal in utero lung development and growth rely upon the expansion of airspaces and the controlled efflux of lung liquid into the amniotic space. Infants ...

仪表近期胎羊多元慢性非麻醉的录音

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胎羔羊子宫内气管压力连续遥测测量

仪表近期胎羊多元慢性非麻醉的录音

摘要

探索更多视频

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基于光纤超声接收机的三维超声针尖跟踪

心率在小鼠中通过无创M型超声心动图测量子宫内

体内 药物对产卵胎颈动脉血流影响的实时研究

微电极阵列技术在完整仔猪脑麻醉诱导神经毒性研究中的应用

围产儿窒息羔羊模型: 新生儿复苏模型的建立

硬膜下软皮质电图 （ECoG） 阵列植入和小型猪的长期皮质记录

围产期小鼠中的非侵入性心电图

在 Fontan 绵羊模型中植入无线遥测设备，用于连续和长期血流动力学监测

意识约束兔子中癫痫发作、心律失常和呼吸暂停的多系统监测

胎羔羊子宫内气管压力连续遥测测量

体内药物对产卵胎颈动脉血流影响的实时研究

硬膜下软皮质电图（ECoG）阵列植入和小型猪的长期皮质记录