The authors would like to thank John Kristensen and S&#248;ren Braad Andersen from the Department of Communication, Aarhus University Hospital, Denmark, for their exceptional help with filming and editing. Animal technician Diana Gyldenl&#248;ve and veterinarian Birgitte Kousgaard, Institute of Clinical Medicine Aarhus University Hospital, Denmark for assisting with animal care. This study was supported by the Lundbeck Foundation, the Laerdal Foundation for Acute Medicine, Central Denmark Region&#8217;s Research Foundation, Augustinus Foundation, Aase and Ejnar Danielsens Foundation, the Institute of Clinical Medicine Aarhus University Hospital, Br&#248;drene Hartmanns Foundation, Karen Elise Jensens Foundation, Fonden til L&#230;gevidenskabens Fremme, and Marie Dorthea og Holger From, Haderslevs Fond.

本方案经丹麦动物实验督察。所有试验动物麻醉整个程序。此协议的复制必须进行按照国家伦理和动物福利的指导方针，并经当地伦理委员会。 1.动物<ol><li>丹麦长白仔猪&lt;24小时岁，体重约1500 - 2000克</li></ol> 2.麻醉和维护液体<ol><li>备所需的麻醉的设备（ 图1）：对于七氟醚给药，酒精棉签，外周静脉导管，橡皮筋，注射器用盐水，丙泊酚（5毫克/千克）的掩模，芬太尼（10微克/千克）和普鲁卡因青霉素（15,000 IU /公斤）。 </li><li>通过提供1-2％的七氟醚通过呼吸面罩诱导麻醉。 <ol><li>通过评估眼睑和提款REFL评估麻醉深度前男友。当确信仔猪被深度麻醉，经皮肤插入外周静脉导管插入耳静脉。 </li><li>以确认外周静脉导管的通畅，冲洗导管用1-2毫升无菌0.9％盐水中。辖推注丙泊酚（5毫克/千克）和芬太尼（10微克/千克）。随后，以推注给药使用1-2毫升无菌的0.9％盐水冲洗该静脉内导管的第二时间。 </li><li>地方注射器异丙酚（10毫克/毫升）和芬太尼（10微克/毫升）在两个分开的注射器注入泵。连接从两个注射泵静脉管到一个三路活塞接合输注到连接到静脉内导管的单行。启动异丙酚连续静脉输注（4-12毫克/千克/小时）和芬太尼（10微克/千克/小时）。一旦连续输注正在运行，不给进一步推注异丙酚和芬太尼。 </li><li>七氟醚气ANEST的中止管理hesia。 </li><li>普鲁卡因注射青霉素（15,000 IU / kg）的皮下或肌肉注射按照抗生素预防局部准则。每天重复。视频显示以sc给药，它在小猪已经显示时相比，即时施用导致更高的血药浓度和更长的半衰期。 </li><li>敷眼润滑剂软膏，以防止眼睛干燥。拉动下眼睑轻轻形成一个口袋状开口。挤少量软膏的口袋里。密切关注分发软膏。检查干燥小时，并根据需要重新申请。 </li><li>发起的5％右旋糖的连续速率输注/ 0.45％的NaCl静脉以10ml / kg /小时。期间和缺氧后降低率到5ml / kg /小时。调节输液速度以保持2-10毫摩尔/升之间的血糖水平。 </li></ol></li></ol><img alt="图1" src="/files/ftp_upload/52454/52454fig1.jpg" /> Figu1.重新对设备和麻醉插管。 <a href="https://www.jove.com/files/ftp_upload/52454/52454fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 3.插管和通气<ol><li>备所需的插管的设备（ 图1）：罗库溴铵（1毫克/千克）为肌肉松弛，绳吊索（绑相同4毫米尼龙绳的两端在一起，形成一个圆， 如图1所示），用于打开口，一个棉签拭子和兽医喉镜直叶片，利多卡因喷雾（100毫克/毫升），用袖带各种尺寸的气管内管（规格3.0毫米，2.5毫米，2.0毫米）500毫升自充气袋（袋阀面罩）通风2毫升注射器的袖口通货膨胀和听诊器。 </li><li>通过插管以下步骤： <ol><li>在仰卧位放置仔猪从每一侧支撑颈部，以确保直喉PAS圣人插管。 </li><li>通过从炉鼻到胸骨切迹（通常约13厘米）的尖端测量估计的气管内管的长度。 </li><li>周围的上颚的地方绳吊带（面朝下），并持有下颚和舌头向上保持口腔开放。 </li><li>辖罗库溴铵（1毫克/千克）静脉诱导肌肉松弛。 </li><li>用喉镜向上抬起舌头。 </li><li>使用棉签拭子释放的仔猪长会厌，它可以是后倾入食道或陷入软腭后面。 </li><li>推进喉镜保持解除对舌根使杓状软骨和声带众目睽睽会厌。 </li><li>应用利多卡因喷雾（100毫克/毫升）局部在喉部，以防止喉痉挛。 </li><li>通过声带推进气管插管。使用旋转运动，以帮助气管狭窄软骨的传球。高级管艾科录制到预先测量的距离，并连接到一个自充气袋（袋阀掩模）用于手动通风。为了帮助减少摩擦插管时喷气管导管与利多卡因喷雾的远端1/3。 </li><li>要确认正确的气管插管位置：为遵守任何迹象显示呼吸困难，ausculate胸部监听双边航空进入两肺，目视确认凝结在气管内管的近端方面存在，并验证呼气末碳的存在二氧化用比色二氧化碳检测器或通过呼气的CO 2读机械呼吸器如果可用上。正常呼气末二氧化碳大约为5％。 2与正常显现波形％一起以上的值将确认气管内导管在气管。 </li><li>充气气管插管袖口，防止误吸。袖带充气压力应小于25厘米水柱2 O，以避免局部缺血损伤<html>到周围的组织。握住气管插管到位，风一块的管周围的磁带，关闭下颚和继续录音周围的吻，以确保管到位。轻轻一拉就管，以确保它保持到位。 </li><li>连接气管插管机械通气。 </li></ol></li><li>调整呼吸机参数：容量控制通气，潮气量（TV）10毫升/千克[或吸气峰压（PIP）15cm宽的压力控制通气。呼气末正压（PEEP）：5厘米。 I：E比为1：2。呼吸频率：35（调整利率维持在4.5-5.5千帕之间呼气末二氧化碳 ）。 </li></ol> 4.监测和体液采取<img alt="图2" src="/files/ftp_upload/52454/52454fig2.jpg" /> 图2.设备进行监控。ANK“&gt;点击此处查看该图的放大版本。 <ol><li>备所需的监测（ 图2）的设备：粘合带，无菌润滑剂，探针连续直肠温度测量，心电图（ECG）电极，脉冲血氧计，剃刀，以及EEG电极。 <ol><li>上一个后腿地方饱和探头，润滑直肠温度探头，并插入6厘米入直肠，放置架空辐射加热器和/或加热可充气的空气床垫，以保持直肠温度在38.5-39℃下的生理水平，和地点心电图电极。 </li><li>将仔猪俯卧位刮胡子1厘米×1厘米皮下针电极脑电图安置区;之一的每个耳朵的前面，并在中线只是眼睛后面的参比电极。清洁电极站点酒精棉签，然后插入电极针皮下。安全电极用胶布和返回仔猪仰卧PO习得。 </li><li>打开aEEG的显示器上。 注：集成脑电图幅度由一个致密的跟踪与上下边距。在这种模式下，上利润率通常是15-50μV，往往在高比婴儿可见。在缺氧它需要注意的是，从心电图神器可能会错误地提升aEEG的痕迹。丸药物（异丙酚或芬太尼），也可能短暂打压aEEG的痕迹，应尽可能避免在实验过程中。药物和临床事件管理应当标注，以方便aEEG的痕迹的解释。 </li></ol></li><li>备所需的放置脐导管中央动脉血压监测和血液采样（ 图3）的设备：无菌手套，无菌披盖，解剖刀，酒精棉签，无菌纸巾，脐静脉导管（5星期五），脐动脉导管（3.5星期五），缝合设定钳，弯微型镊子，剪刀，持针器缝合（例如 ，尺寸3-0），透明粘合贴剂，将5ml注射器采血。 </li></ol><img alt="图3" src="/files/ftp_upload/52454/52454fig3.jpg" /> 图3.设备脐带线和血液采样。 <a href="https://www.jove.com/files/ftp_upload/52454/52454fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol start="3"><li>消毒和悬垂性肚脐周围的区域。用手术刀切割脐带作为接近皮肤越好。如果不公开脐部血管麻醉的仔猪，剪脐带蒙皮2毫米脐带下方，露出血管。确定的两个小脐动脉和所述一个较大脐静脉（ 图4）。 </li><li>用弯微型镊子扩张动脉（ 图4），然后将脐动脉导管（3.5 FR）。估计插入长度厘米至3×体重（kg）+ 10（基于正确放置在降主动脉上面肾动脉尸检经验公式）。 </li><li>放置第二个5星期五导管5厘米的脐静脉（ 图4）。验证血管内导管放置由血液回流。安全导管放置一个荷包缝合脐部周围，绕过每一个脐导管缝合结束，打结。导管覆盖有透明胶粘敷料。 </li><li>收集血液样品在预先指定的时间点：1）紧接缺氧前，2）30分钟到缺氧损伤3）在45分钟缺氧损伤4）缺氧损伤后2小时的末端。取决于血样实验其他时间点的目的而被选择。 </li><li>从使用缺氧时的血液样本动脉血气分析，以验证所造成的缺血缺氧（表1）血气变化。需要注意的是仔猪具有低血红蛋白牛逼出生（约8克/分升），并可能成为频繁采血贫血。撤回小于2毫升血液/千克拉伸体重，降到低于5毫升血液/ kg体重24小时内并附着到用于血液取样本地指南。贫血的症状包括血细胞比容下降和心动过速。 </li><li>动脉线连接到显示器持续动脉内血压监测（MABP）。使用静脉线液体和药物管理局。 </li></ol><img alt="图4" src="/files/ftp_upload/52454/52454fig4.jpg" /> 图4.脐船只。脐静脉（右）和两个脐动脉（左）之一。 <a href="https://www.jove.com/files/ftp_upload/52454/52454fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 5.缺氧<ol><li>等待完成后监测60分钟。通过切换到4％O 2缺氧诱导</suB&gt; / 96％N 2气混合物。监控重要参数和aEEG的密切合作，继续缺氧45分钟。 </li><li>一旦aEEG的轨迹是平面（上缘&lt;7μV），通过改变吸入氧分数（氧合指数2）调整氧合和平均气道压力的最高氧合指数2水平保持平坦跟踪aEEG的根据图5所示的流程图。瞄准MABP低于基线的70％，持续至少10分钟，并在需要时降低氧合指数2，以确保低血压和缺血。低血压的描述水平以前已经显示由他人19,13,30以产生有关缺氧缺血性脑损伤。 </li><li>在严重的情况下，低血压（MABP &lt;25毫米汞柱）治疗逐步如下：暂时增加2氧合指数由1-5％，推注生理盐水（10毫升/公斤），输注多巴胺（5-20 ​​微克/公斤/分钟），和去甲肾上腺素输液（20 NG-1微克/千克/分钟）。 </li><li>若发作持续&gt; 10分钟的（阵挛，补虚Øř肌阵挛发作，通常局灶性或通过在aEEG的振幅突然变化）所指示治疗阶段性地（允许30分钟在进行到下一个注射）之前：苯巴比妥静脉20毫克/公斤的缓慢推注，重复巴比妥静脉20毫克/公斤，并咪达唑仑0.5 IV毫克/千克。 <ol><li>如果发作开发和无响应药物治疗安乐死表示。 </li></ol></li></ol><img alt="图5" src="/files/ftp_upload/52454/52454fig5.jpg" /> 图5.缺氧缺血性流程图。流程图显示氧合调整（F I O 2和P AW）根据aEEG的响应。平均气道压力（P AW）通过改变PIP / TV（PIP较低给人低磷AW）和呼吸频率调整（降低RR给人低磷AW）。针对aEEG的=上一丝缘&lt;5μV和跟踪下缘&gt; 3μV（平均4μV）。目标心脏率（HR）=&gt; 80.目标平均动脉压（MABP）= MABP&gt; 25。 <a href="https://www.jove.com/files/ftp_upload/52454/52454fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 6. 72小时生存<ol><li>监测缺氧损伤后的仔猪紧密结合，并逐步减少输注丙泊酚和芬太尼的速度。拔管在该点处的动物自愿呼吸。 </li><li>在生存期，保持仔猪24小时观看动物设施由受过培训的重症监护室和监控人员。四，给予输注葡萄糖以20ml /公斤每小时2-3。在后缺氧缺血部分奶瓶喂养26-48小时可以启动。吸入气管和肺部可以通过奶瓶喂养，如果出现的呕吐反射不存在。 </li><li>如果相关的实验中，使用由托勒森等人开发的评分系统评价神经学状况，在所述详细的原始出版物17。 </li></ol> 7.成果评价<ol><li> 72小时后，麻醉并在第2节和第3执行相关的成像方式所描述的Munkeby 等 31等2 MRI扫描再打开门窗通风。 </li><li>在实验结束时安乐死仔猪​​用致死剂量的戊巴比妥（5克/千克四）。 </li><li>根据实验的目的制备脑进行检查。 <ol><li>对福尔马林固定的组织的脑组织学：使用心脏灌注用PBS中的4％多聚甲醛，接着解剖和后固定在多聚甲醛如Robertson 等人的 2，Chakkarapani 等 1或Liu 等人 18或取出脑和所描述的安德烈森等浸入4％多聚甲醛。32 </li><li>对于需要分析单元冷冻的组织（ 例如 ，RNA分析或酶活性试验33）：取出脑，解剖出感兴趣的大脑区域和扣冻结的最大1厘米组织块×1cm的在液氮中所描述的Munkeby 等人 33 </li></ol></li></ol>

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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+survival+model+of+hypoxia/ischaemia+in+the+piglet+suitable+for+neuroprotection+studies.">An improved survival model of hypoxia/ischaemia in the piglet suitable for neuroprotection studies.</a> Brain research. 919, 122-131 (2001).</li><li>LeBlanc, M. H., Li, X. Q., Huang, M., Patel, D. M., Smith, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMPA+antagonist+LY293558+does+not+affect+the+severity+of+hypoxic-ischemic+injury+in+newborn+pigs.">AMPA antagonist LY293558 does not affect the severity of hypoxic-ischemic injury in newborn pigs.</a> Stroke; a journal of cerebral circulation. 26, 1908-1914 (1995).</li><li>Andresen, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nicotine+affects+the+expression+of+brain-derived+neurotrophic+factor+mRNA+and+protein+in+the+hippocampus+of+hypoxic+newborn+piglets.">Nicotine affects the expression of brain-derived neurotrophic factor mRNA and protein in the hippocampus of hypoxic newborn piglets.</a> J Perinat Med. 37, 553-560 (2009).</li><li>Cheung, P. Y., Gill, R. S., Bigam, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+swine+model+of+neonatal+asphyxia.">A swine model of neonatal asphyxia.</a> Journal of visualized experiments : JoVE. , (2011).</li><li>Thoresen, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+piglet+survival+model+of+posthypoxic+encephalopathy.">A piglet survival model of posthypoxic encephalopathy.</a> Pediatric research. 40, 738-748 (1996).</li><li>Liu, X., Tooley, J., Loberg, E. M., Suleiman, M. S., Thoresen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immediate+hypothermia+reduces+cardiac+troponin+I+after+hypoxic-ischemic+encephalopathy+in+newborn+pigs.">Immediate hypothermia reduces cardiac troponin I after hypoxic-ischemic encephalopathy in newborn pigs.</a> Pediatric research. 70, 352-356 (2011).</li><li>Bjorkman, S. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxic/Ischemic+models+in+newborn+piglet:+comparison+of+constant+FiO2+versus+variable+FiO2+delivery.">Hypoxic/Ischemic models in newborn piglet: comparison of constant FiO2 versus variable FiO2 delivery.</a> Brain research. 1100, 110-117 (2006).</li><li>Robertson, N. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Which+neuroprotective+agents+are+ready+for+bench+to+bedside+translation+in+the+newborn+infant.">Which neuroprotective agents are ready for bench to bedside translation in the newborn infant.</a> The Journal of pediatrics. 160, 544-552 (2012).</li><li>Jacobs, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cooling+for+newborns+with+hypoxic+ischaemic+encephalopathy.">Cooling for newborns with hypoxic ischaemic encephalopathy.</a> Cochrane Database Syst Rev. 1, CD003311 (2013).</li><li>Andresen, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resuscitation+with+21+or+100%+oxygen+in+hypoxic+nicotine-pretreated+newborn+piglets:+possible+neuroprotective+effects+of+nicotine.">Resuscitation with 21 or 100% oxygen in hypoxic nicotine-pretreated newborn piglets: possible neuroprotective effects of nicotine.</a> Neonatology. 93, 36-44 (2008).</li><li>Karlsson, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delayed+hypothermia+as+selective+head+cooling+or+whole+body+cooling+does+not+protect+brain+or+body+in+newborn+pig+subjected+to+hypoxia-ischemia.">Delayed hypothermia as selective head cooling or whole body cooling does not protect brain or body in newborn pig subjected to hypoxia-ischemia.</a> Pediatric research. 64, 74-78 (2008).</li><li>Kovacs, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asphyxia-induced+release+of+alpha-melanocyte-stimulating+hormone+in+newborn+pigs.">Asphyxia-induced release of alpha-melanocyte-stimulating hormone in newborn pigs.</a> Peptides. 22, 1049-1053 (2001).</li><li>Varvarousi, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asphyxial+cardiac+arrest,+resuscitation+and+neurological+outcome+in+a+Landrace/Large-White+swine+model.">Asphyxial cardiac arrest, resuscitation and neurological outcome in a Landrace/Large-White swine model.</a> Laboratory animals. 45, 184-190 (2011).</li><li>Tammela, O., Pastuszko, A., Lajevardi, N. S., Delivoria-Papadopoulos, M., Wilson, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity+of+tyrosine+hydroxylase+in+the+striatum+of+newborn+piglets+in+response+to+hypocapnic+hypoxia.">Activity of tyrosine hydroxylase in the striatum of newborn piglets in response to hypocapnic hypoxia.</a> Journal of neurochemistry. 60, 1399-1406 (1993).</li><li>Cote, A., Barter, J., Meehan, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-dependent+metabolic+effects+of+repeated+hypoxemia+in+piglets.">Age-dependent metabolic effects of repeated hypoxemia in piglets.</a> Canadian journal of physiology and pharmacology. 78, 321-328 (2000).</li><li>Tichauer, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+the+severity+of+perinatal+hypoxia-ischemia+in+piglets+using+near-infrared+spectroscopy+to+measure+the+cerebral+metabolic+rate+of+oxygen.">Assessing the severity of perinatal hypoxia-ischemia in piglets using near-infrared spectroscopy to measure the cerebral metabolic rate of oxygen.</a> Pediatric research. 65, 301-306 (2009).</li><li>Jasani, M. S., Salzman, S. K., Tice, L. L., Ginn, A., Nadkarni, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anesthetic+regimen+effects+on+a+pediatric+porcine+model+of+asphyxial+arrest.">Anesthetic regimen effects on a pediatric porcine model of asphyxial arrest.</a> Resuscitation. 35, 69-75 (1997).</li><li>Chakkarapani, E., Thoresen, M., Liu, X., Walloe, L., Dingley, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenon+offers+stable+haemodynamics+independent+of+induced+hypothermia+after+hypoxia-ischaemia+in+newborn+pigs.">Xenon offers stable haemodynamics independent of induced hypothermia after hypoxia-ischaemia in newborn pigs.</a> Intensive care medicine. 38, 316-323 (2012).</li><li>Munkeby, B. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+piglet+model+for+detection+of+hypoxic-ischemic+brain+injury+with+magnetic+resonance+imaging.">A piglet model for detection of hypoxic-ischemic brain injury with magnetic resonance imaging.</a> Acta radiologica. 49, 1049-1057 (2008).</li><li>Andresen, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Newborn+piglets+exposed+to+hypoxia+after+nicotine+or+saline+pretreatment:+long-term+effects+on+brain+and+heart.">Newborn piglets exposed to hypoxia after nicotine or saline pretreatment: long-term effects on brain and heart.</a> J Matern Fetal Neonatal Med. 22, 161-168 (2009).</li><li>Munkeby, B. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resuscitation+with+100%+O2+increases+cerebral+injury+in+hypoxemic+piglets.">Resuscitation with 100% O2 increases cerebral injury in hypoxemic piglets.</a> Pediatric research. 56, 783-790 (2004).</li></ol>

作者什么都没有透露。

由于它的复杂性，所描述的模型仅可在设施认证实施​​并经历了动物实验。批准当地伦理委员会之前必须对实验的起始获得，并且最佳动物福利必须保证在任何时候。由于模型是基于试验动物的存活，但重要的是无菌的环境是在侵入性过程，以防止感染维持。 麻醉的选择是很重要的，因为大多数如果不是所有的麻醉药物具有潜在神经保护性质。在先前公布的模型之间的差?...

围产期窒息与缺氧缺血性脑病（HIE）相关的急性和不可预测的频繁条件。该协议的总体目标是展示围产期缺氧缺血性脑病的仔猪存活率模型。该模型可用于研究对体液如血液，脑脊液和尿液的实验处理对神经病理学，磁共振成像和光谱学（MRI和MRS）和生物标志物的新生儿脑和不同程度缺氧缺血的效果。该模型也证明用于研究心血管系统，呼吸系统，肾和肝，所有这些都受到影响全球缺氧缺血有用。 围产期窒息是妥协供氧产或立即产后期的结果。产缺氧事件，占全球的66万死亡人数每年，大约有四分之一的世界2.9磨离子新生儿死亡，2012年3。2010年115万婴儿估计已经开发新生儿脑病出生后窒息4。 HIE定义为脑病妊娠34周后出生的婴儿发生在1-3 / 1000个活产5在工业化世界和高达8.5 / 1000名活产婴儿在发展中国家4。死亡的风险为10-60％，和神经障碍中的幸存者30-100％6,7-风险。 5020万伤残调整生命年（DALYs）是由于分娩缺氧事件4。目前唯一的治疗比为支持其他HIE是后缺氧低温。因此，在诊断过程和治疗策略的进步所必需的改进的HIE 8的管理。 在围产期窒息及新生儿脑损伤后管理预后改进基于扩大潜在疾病机制的知识ð可能的治疗方法。缺氧缺血性脑病的动物模型中是特别有用的为不同的临床事件可能导致HIE和发病率中的任何单个分娩中心是低5。测试新的预后和诊断工具和治疗策略时的实验装置，其中生物变化的影响可被最小化，是必不可少的。动物模型应接近临床情况尽可能接近，从而有利于诱导损伤和参与该疾病和it&#39;s结果9的动态过程背后的病理机制的理解。新生儿缺氧缺血性脑病的动物模型中已经包括了若干种，包括啮齿动物，羊和猪。相比较而言，新生仔猪具有较高的相似性人类新生儿相对于大小，心血管系统10和脑的成熟在交货11,12的时间。监控，仪器仪表和成果评价中的仔猪模型类似于THAT在临床护理婴儿缺氧缺血性脑病的使用。因此，存在一个高度翻译成新生儿护理从这个模型。 围产期缺氧，缺氧缺血性脑病仔猪模型所使用的许多团体和不同的许多领域13。根据实验的目的，必须小心注意支付给药物的选择，诱导缺血缺氧，控制侮辱持续时间和严重程度，后期侮辱复苏和护理，成果评价方法的方法。为了避免偏见的随机试验的设计应始终在干预研究中使用。 当诱导缺氧缺血性损伤所采用的方法是很重要的。全球缺氧导致的HIE经常导致多器官衰竭涉及脑，心脏，肺，肾和肝。根据评估结果，缺氧缺血性脑病模型应该基于全球缺氧缺血，而不是依赖于脑缺血， 例如 ，开车结扎otid动脉14。最近的一篇论文缺氧应用颈动脉受压的组合（2氧合指数12％），并同时维持平均动脉压“40毫米汞柱2。另一组引起的全球缺氧8％的O 2，直到负碱过量&gt; 20毫摩尔/升或平均动脉血压（MABP）&lt;15毫米汞柱，并处死动物在4小时15。缺氧也被滴定通过心输出量（基线的30％-40％），MABP（30-35毫米汞柱）和动脉pH值（6.95-7.05）16。 全球缺氧缺血aEEG的抑制一个类似于本报告中提出滴定，车型已经证明了脑病是临床，电生理，神经病理学和媲美的窒息足月儿17,18中发现的情况。 缺氧缺血性脑病引起的程度是至关重要的。 HIE的有用的动物模型还必须考虑新的测试直径诺斯替过程和治疗方案。为了实现这一目标，该车型应该引起中度HIE，其中有一个潜在的治疗为重型颅脑损伤，很少或没有处理潜在的评估新的治疗方法的时候会少有关。对缺氧的耐受试验动物之间的差别很大。以前的研究已经表明，更一致的脑损伤，可以实现，并且更多的动物存活17,19通过使用在整个一组氧合指数2的值根据每个仔猪由振幅整合脑电图（aEEG的）来评价脑响应个体化的诱导缺氧而不是缺氧事件。 aEEG的抑制的持续时间关联到脑损伤的程度，以在几个组织病理学改变&lt;20分钟aEEG的抑制和严重癫痫发作是增加&gt; 45分钟aEEG的抑制。对HIE神经保护治疗最近的审查中发现的需要生存模式，使行为结果MEAS数目字的动物模型20。 还有的提出HIE仔猪模型的许多优点。它是基于一个物种，其中结果是高度可能转化为人体生理学。全球缺氧缺血模型多器官衰竭和缺血缺氧的滴定通过aEEG的诱导一致的程度脑损伤与存活的临床相关的结果，使得生物标志物，MRI和行为可以在相关的时间点进行评价。 围生期窒息和缺氧缺血性脑病仔猪模型不仅当前洞察HIE病理生理显著贡献，而且还成功之前的临床试验，最终导致人类的新疗法。仔猪模型研究起到在建立低温作为HIE 21处理了关键作用，并且在新生儿复苏研究22被使用。执行中窒息和缺氧缺血性脑病，是时候研究不同群体已经使用仔猪模型ð研究包括低温23，α-黑素细胞激素24，心跳骤停25，酪氨酸羟化酶活性26，反复低氧暴露27，NMDA受体活性14，和近红外光谱28。 仔猪HIE模型本报告中提出的在技术上具有挑战性的工作，作为程序的过程中微小的调整，可能会导致要么太轻微或太严重颅脑损伤29.2。我们发现，现有文献缺乏足够的细节重现此前公布​​的模型。因此，我们在这里展示所必需的这份报告在建立仔猪72小时的生存模式的技术程序的每一步，使研究人员能够建立这种先进的型号为缺氧缺血性脑病的研究。

<table border="0" cellpadding="0" cellspacing="0" width="1034">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:647px;">Warm-touch-pediatric blanket</td>
			<td style="width:211px;">Covidien</td>
			<td style="width:176px;">5030840</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adhesive Apertrue Drape</td>
			<td>Barrier</td>
			<td>915447</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Utility Drape (sterile) 75x80 cm</td>
			<td>Barrier</td>
			<td>800530</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neoflon</td>
			<td>BD - Luer</td>
			<td>391350</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:647px;">Laryngoscope</td>
			<td style="width:211px;">Miller</td>
			<td style="width:176px;">85-0045</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Endotracheal tube 2.5 mm&#160;</td>
			<td>Covidien</td>
			<td>111-25</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Endotracheal tube 3.0 mm with cuff</td>
			<td>Unomedical</td>
			<td>MM61110030</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Endotracheal tube 3.5 mm with cuff</td>
			<td>Unomedical</td>
			<td>MM61110035</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:647px;">Anesthesia machine</td>
			<td style="width:211px;">GE Healthcare</td>
			<td style="width:176px;">1009-9002-000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EEG - electrodes/disposable subdermal needle electrode</td>
			<td>Cephalon</td>
			<td>ACCE120550</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ECG - electrodes</td>
			<td>medtronic</td>
			<td>3010107-003</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ECG-electrodes for MR</td>
			<td>philips</td>
			<td>ACCE120550</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Arterial blood sampler - aspirator</td>
			<td>Radiometer medical ApS</td>
			<td>956552</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polyurethane Umbilical vein catheter (5 Fr/Ch)</td>
			<td>Covidien</td>
			<td>8888160341</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polyurethane Umbilical vein catheter (3,5 Fr/ch)</td>
			<td>Covidien</td>
			<td>8888160333</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Suture set (size 3-0)</td>
			<td>Covidien</td>
			<td>8886 623341</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD Spinal needle 0.7x38mm</td>
			<td>BD needles</td>
			<td>405254</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:647px;">Gas with 96% Nitrogen / 4% oxygen</td>
			<td style="width:211px;">Air Liquide</td>
			<td style="width:176px;">made on order</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:647px;">NeuroMonitor (CFM) system</td>
			<td>Natus Medical Incorporated</td>
			<td>OBM70002</td>
		</tr>
	</tbody>
</table>

a piglet model of neonatal hypoxic-ischemic encephalopathy

出生窒息，从而导致缺氧缺血性脑病（HIE），占全球的66万例死亡，每年，大约有四分之一的世界290万新生儿死亡。缺氧缺血性脑病的动物模型有助于在HIE的病理生理的理解，并强调，在发生脑损伤的动态过程，由于围产期窒息。因此，动物的研究表明一个时间窗后的损伤的治疗策略。低温已经过测试作为pdiglet模型HIE处理，并随后在临床试验中证明是有效的。该模型的变化在辅助神经保护方法和氙气和褪黑激素的仔猪研究的研究已应用于导致临床I期和II期试验1,2。仔猪HIE模型还用于新生儿resuscitation-和血流动力学研究，以及脑缺氧细胞水平上的调查。然而，这是一个在技术上具有挑战性模型和变化的协议，可能会导致要么太轻微或太严重的脑损伤。在这篇文章中，我们表现出必要建立新生儿缺氧缺血性脑病稳定仔猪模型的技术方法。首先，新生仔猪（&lt;24小时的时候，平均重量1500克）麻醉，插管，并在设置相媲美，在新生儿重症监护病房发现监控。全球缺氧缺血通过降低吸入氧分数达到全球缺氧，低血压，通过缺血和一台跟踪振幅整合脑电图（aEEG的）指示脑缺氧所致。生存是通过根据所述aEEG的响应和血压调节充氧促进。脑损伤通过组织病理学和磁共振成像72小时后定量。

缺血缺氧对大脑的损伤引起的过程中发生的影响记录在案记录aEEG的痕迹。代表性aEEG的跟踪示于图6。 <img alt="图6" src="/files/ftp_upload/52454/52454fig6.jpg" /> 图6.代表aEEG的痕迹。低幅度因缺血缺氧。 <a href="https://www.jove.com/files/ftp_upload/52454/52454fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> p...

Watch this Scientific Journal Video about A Piglet Model of Neonatal Hypoxic-Ischemic Encephalopathy at JoVE.com

A Piglet Model of Neonatal Hypoxic-Ischemic Encephalopathy

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

新生儿缺氧缺血性脑病的小猪模型

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

a-piglet-model-of-neonatal-hypoxic-ischemic-encephalopathy

Research

JoVE Journal

Medicine

19.5K 次观看.  Institute of Clinical Medicine, Aarhus University Hospital. Hypoxic-ischemic encephalopathy following perinatal asphyxia can be studied using animal models. We demonstrate the procedures necessary for establishing a piglet model of neonatal hypoxic-ischemic encephalopathy. 

视频: A Piglet Model of Neonatal Hypoxic-Ischemic Encephalopathy

A Swine Model of Neonatal Asphyxia

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

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

新生儿窒息了猪模型

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

科研

医学

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

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) has a lifetime prevalence of 14% and is the most common urological diagnosis for men under the age of 50, yet it is the least understood and studied chronic pelvic pain disorder. A significant subset of patients with chronic pelvic pain report having experienced early life stress or abuse, which can markedly affect the functioning and regulation of the hypothalamic-pituitary-adrenal (HPA) axis. Mast cell activation, which has been shown to be increased in both urine and expressed prostatic secretions of CP/CPPS patients, is partially regulated by downstream activation of the HPA axis. Neonatal maternal separation (NMS) has been used for over two decades to study the outcomes of early life stress in rodent models, including changes in the HPA axis and visceral sensitivity. Here we provide a detailed protocol for using NMS as a preclinical model of CP/CPPS in male C57BL/6 mice. We describe the methodology for performing NMS, assessing perigenital mechanical allodynia, and histological evidence of mast cell activation. We also provide evidence that early psychological stress can have long-lasting effects on the male urogenital system in mice.

We are measuring perigenital mechanical sensitivity and mast cell activation in the prostate of male C57BL/6 mice that underwent an early life stress paradigm &#8211; neonatal maternal separation, in order to induce a preclinical model of chronic prostatitis/chronic pelvic pain syndrome.

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

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

A Neonatal Mouse Spinal Cord Compression Injury Model

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

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

新生儿小鼠脊髓压迫损伤模型

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

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

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

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

汽车测试的脑瘫新生儿小鼠模型电池

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

Myocardial Infarction in Neonatal Mice, A Model of Cardiac Regeneration

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

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

心肌梗死新生小鼠，心肌再生的模型

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

Use of a Piglet Model for the Study of Anesthetic-induced Developmental Neurotoxicity (AIDN): A Translational Neuroscience Approach

Anesthesia cannot be avoided in many cases when surgery is required, particularly in children. Recent investigations in animals have raised concerns that anesthesia exposure may lead to neuronal apoptosis, known as anesthesia-induced developmental neurotoxicity (AIDN). Furthermore, some clinical studies in children have suggested that anesthesia exposure may lead to neurodevelopmental deficits later in life. Nonetheless, an ideal animal model for preclinical study has yet to be developed. The neonatal piglet represents a valuable model for preclinical study, as they share a striking number of developmental similarities with humans.
The anatomy and physiology of piglets allow for implementation of rigorous human perioperative conditions in both survival and non-survival procedures. Femoral artery catheterization allows for close monitoring, thus enabling prompt correction of any deviation of the piglet's vital signs and chemistries. In addition, there are multiple developmental similarities between piglets and human neonates. The techniques required to use piglets for experimentation will require experience to master. A pediatric anesthesiologist is a critical member of the investigative team. We describe, in a general sense, the appropriate use of a piglet model for neurodevelopmental study.

Use of a Piglet Model for the Study of Anesthetic-induced Developmental Neurotoxicity AIDN: A Translational Neuroscience Approach

Anesthesia-induced developmental neurotoxicity (AIDN) research has focused on rodents, which are not broadly applicable to humans. Non-human primate models are more relevant, but are cost-prohibitive and difficult to use for experimentation. The piglet, in contrast, is a clinically relevant, practical animal model ideal for the study of anesthetic neurotoxicity.

使用仔猪模型研究麻醉诱发的发育神经毒性（AIDN）：一种转化神经科学方法

Anesthesia cannot be avoided in many cases when surgery is required, particularly in children.  Recent investigations in animals have raised concerns that ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

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

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

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

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

A Neonatal BALB/c Mouse Model of Necrotizing Enterocolitis

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

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

一种新生儿BALB/c坏死性小肠结肠炎小鼠模型

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

Effect of Hyaluronic Acid 35 kDa on an In Vitro Model of Preterm Small Intestinal Injury and Healing Using Enteroid-Derived Monolayers

In vitro scratch wound assays are commonly used to investigate the mechanisms and characteristics of epithelial healing in a variety of tissue types. Here, we describe a protocol to generate a two-dimensional (2D) monolayer from three-dimensional (3D) non-human primate enteroids derived from intestinal crypts of the terminal ileum. These enteroid-derived monolayers were then utilized in an in vitro scratch wound assay to test the ability of hyaluronan 35 kDa (HA35), a human milk HA mimic, to promote cell migration and proliferation along the epithelial wound edge. After the monolayers were grown to confluency, they were manually scratched and treated with HA35 (50 &#181;g/mL, 100 &#181;g/mL, 200 &#181;g/mL) or control (PBS). Cell migration and proliferation into the gap were imaged using a transmitted-light microscope equipped for live-cell imaging. Wound closure was quantified as percent wound healing using the Wound Healing Size Plugin in ImageJ.&#160;The scratch area and rate of cell migration and the percentage of wound closure were measured over 24 h. HA35 in vitro accelerates wound healing in small intestinal enteroid monolayers, likely through a combination of cell proliferation at the wound edge and migration to the wound area. These methods can potentially be used as a model to explore intestinal regeneration in the preterm human small intestine.

Effect of Hyaluronic Acid 35 kDa on an In Vitro Model of Preterm Small Intestinal Injury and Healing Using Enteroid-Derived Monolayers

This protocol describes a method to establish and perform a scratch wound assay on two-dimensional (2D) monolayers derived from three-dimensional (3D) enteroids isolated from non-human primate ileum.

透明质酸35 kDa对肠源单层早产小肠损伤体 外 模型及愈合的影响

In vitro scratch wound assays are commonly used to investigate the mechanisms and characteristics of epithelial healing in a variety of tissue types. ...

In Vitro Apical-Out Enteroid Model of Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is a devastating disease affecting preterm infants, characterized by intestinal inflammation and necrosis. Enteroids have recently emerged as a promising system to model gastrointestinal pathologies. However, currently utilized methods for enteroid manipulation either lack access to the apical surface of the epithelium (three-dimensional [3D]) or are time-consuming and resource-intensive (two-dimensional [2D] monolayers). These methods often require additional steps, such as microinjection, for the model to become physiologically translatable. Here, we describe a physiologically relevant and inexpensive protocol for studying NEC in vitro by reversing enteroid polarity, resulting in the apical surface facing outward (apical-out). An immunofluorescent staining protocol to examine enteroid barrier integrity and junctional protein expression following exposure to tumor necrosis factor-alpha (TNF-&#945;) or lipopolysaccharide (LPS) under normoxic or hypoxic conditions is also provided. The viability of 3D apical-out enteroids exposed to normoxic or hypoxic LPS or TNF-&#945; for 24 h is also evaluated. Enteroids exposed to either LPS or TNF-&#945;, in combination with hypoxia, exhibited disruption of epithelial architecture, a loss of adherens junction protein expression, and a reduction in cell viability. This protocol describes a new apical-out NEC-in-a-dish model which presents a physiologically relevant and cost-effective platform to identify potential epithelial targets for NEC therapies and study the preterm intestinal response to therapeutics.

In Vitro Apical-Out Enteroid Model of Necrotizing Enterocolitis

This protocol describes an apical-out necrotizing enterocolitis (NEC)-in-a-dish model utilizing small intestinal enteroids with reversed polarity, allowing access to the apical surface. We provide an immunofluorescent staining protocol to detect NEC-related epithelial disruption and a method to determine the viability of apical-out enteroids subjected to the NEC-in-a-dish protocol.

体外 坏死性小肠结肠炎的顶端出肠样模型

Necrotizing enterocolitis (NEC) is a devastating disease affecting preterm infants, characterized by intestinal inflammation and necrosis. Enteroids have ...