We would like to give a big thanks to the Visible Heart Laboratory staff and students that have helped with this project: Nate Menninga, Lars Mattison and Megan Schmidt.

该协议被批准明尼苏达机构动物护理和使用委员会的大学。 <img alt="图1" src="/files/ftp_upload/52600/52600fig1.jpg" /> 图1图研究范式的 <a href="https://www.jove.com/files/ftp_upload/52600/52600fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 1.手术猪的制备<ol><li>辖5-7mg / kg的替来他明/ zilazipam的通过肌肉注射猪（70-80公斤）。将耳静脉导管，并通过静脉通道给予5-7毫克/千克美索比妥。插管与气管插管和通风的动物。 </li><li>递送异氟烷&gt; 1.2％的MAC在室内空气和氧气的混合物维持麻醉的手术平面。确保动物在阿耐思特的深面hesia之前，通过检查没有下巴基调，保持最低肺泡浓度高于麻醉猪的任何操作。最初的麻醉深度检查，通过确保无颚音，异氟醚浓度进行的整个过程中（&gt; 1.5 MAC）持续监测，以确保麻醉深面。 </li><li>小心访问与烧灼和钝性分离右颈外和颈动脉。放置一个8.5F Swan-Ganz导管在颈外和膨胀的气球至1.5立方厘米。然后通过右心房穿越，进入心室，并通过肺动脉瓣下肺动脉直到感觉到楔压。 </li><li>放气该气囊和离开所述导管在这个位置。记录使用气球右心房压力和肺动脉压力。此外，记录在肺楔压或压力（或者如果肺部被认为是正常的，则还记录留下了试验压力）。 注意:此导管还允许心输出量的估计。 </li><li>放置一个附加5.0F气球压力导管在颈外并进入右心室连续记录压力。 </li><li>放置在颈动脉中的5.0F气球压力导管，脱出通过主动脉瓣，并在左心室维护。刷新所有压力导管连接到压力换能器20单位/毫升heparanized盐水，并记录从左心室所有的压力数据，右心室（和估计的心输出量），肺动​​脉压和右心房压。 </li><li>对于外围监控，通过将一个导管18F为实时血压，血气监测进入股动脉的分支。在手术过程中记录心脏速率和温度。 </li><li>小心访问与烧灼和钝性分离左侧颈外静脉。将两个11.0F止血引荐的枸骨拉尔安全与0丝线缝合。将导引导管的介绍人。 </li><li>下透视引导，地方主动固定在右心耳和右心室的心尖引线。 注:固定引线，心脏连接到心脏起搏器植入手术植入电线。 </li><li> Connectanalyzer电缆引线并测试利用程序员捕获。设置参数为8V，0.25毫秒的持续时间，并且在最少的比当时的固有速率高10 BPM设置起搏速率。记录为每根引线的相对阻抗。 </li><li>使从剑突内侧外科手术切口的胸锁乳突肌的插入附近。使用胸骨锯，除去从胸骨（龙骨骨）的前突起，以使胸骨的部分内侧清扫。使用通过胸骨骨结构的剩余部分切成一剪切力。解剖胸骨，心包韧带和收回sternu米</li><li>使用钝性分离至心包从胸腔里料分开。使在心包内侧矢状切口（3-5厘米），并在每个角创建具有四方形结缝线一个心包支架（右，左，上，下的网站）。 </li><li>将临时双极率先左心室心尖区放置一个单极暴跌临时起搏导线插入左心耳。 </li><li>安乐死是通过去除深麻醉下的心脏完成的。 </li></ol> 2.筹备电生理监测<ol><li>心脏定位系统。 <ol><li>选择在心脏标测系统的AF研究连接室心外膜双极起搏导线和心外膜导线与鳄鱼夹在接线盒适当的设置心耳。连接心电图和接地焊盘到系统。 </li></ol></li><li>示波器和草刺激。 <ol><li>连接双极左心室起搏导线和单极心房率先突围框连接到电气验证示波器一草刺激。 </li></ol></li><li>程控电刺激。 <ol><li>固定右心房导线和右心室导线的起搏器放置程序员遥测头像放在了心脏起搏器。选择起搏电极刺激（PES）的研究，并设置参数8V;删除所有患者参数。设置起搏持续时间在400毫秒（或300毫秒）设定为8个脉冲。 </li></ol></li></ol> 3. 在原位研究<ol><li>不应期:研究范式。 <ol><li>确定心房不应期（AERP），心房心室节点不应期（AVNERP）和基线心室有效不应期（VERP），并在30分钟和60分钟后化合物/药物/对照处理的心包输送;额外的时间点可以被添加为德IRED。 </li></ol></li><li>不应期:编程器设置。 <ol><li>突发诱导PES研究参数8步（400毫秒或300毫秒），其次是最低300毫秒起搏。降低起搏直到心室/心房（分别）无法收缩。这是从起搏引线的传感说明;从而确定相对不应期。 </li></ol></li></ol><img alt="图2" src="/files/ftp_upload/52600/52600fig2.jpg" /> 图2. EnSiteSystem:AERP，AVNERP和VERP测定 <a href="https://www.jove.com/files/ftp_upload/52600/52600fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol start="3"><li>参数:相对AF负荷的测定。 <ol><li>维持动物的心脏房颤，1分钟后，允许它继续留在AF长达10分钟: 即在5焦耳震惊直接拨片心房终止AF之前。萨尔瓦多ICIT AF感应到在每个时间点的10倍。请注意，在本研究中。 AF定义为缺乏P波的不稳定节奏。 </li></ol></li><li> AF负担:草刺激和示波器设置。 <ol><li>优化基层刺激的参数有2秒的脉冲持续时间通过急跌起搏导线左心耳（左心耳）提供4赫兹;通过标准的示波器验证这一点。递送单脉冲到LAA诱导的AF。 </li></ol></li></ol> 4.心外植，复苏（移植模型） <ol><li>主动脉根部插管。 <ol><li>仔细解剖周围的升主动脉的心包组织，取出2.0 Ethibond缝合pericardium.Suture两个点（2-3厘米除外）在升主动脉和管理30000台肝素静脉注射。 </li><li>放置缝合点之间的主动脉根部套管和固定主动脉。与加压输送系统，除去叔他口针斜面放置一个夹在插管不允许流量。 </li></ol></li><li>搏准备。 <ol><li>辖150毫米/ Hg的压力下通过加压袋冷圣托马斯心麻痹液进入升主动脉。固定附加到加压停跳与远端的3路活栓的冲洗导管。固定止水栓到主动脉根部插管，通过导管释放停跳的稳定冲洗，并固定在主动脉根部插管。 </li></ol></li><li>交叉夹紧。 <ol><li>夹紧下腔静脉与atraumaticclamp 1-2厘米只是优于隔膜。钳与升主动脉优于主动脉根插管无创伤角度营1-2厘米升主动脉。 </li></ol></li><li>跨钳/搏。 <ol><li>从主动脉根部套管除去夹子，在150毫米/汞柱朝向主动脉瓣冲洗停跳，关闭主动脉瓣和perfusING冠状动脉。 </li><li>钳上腔静脉，并进行小切口在肺动脉，以免过度加压的心脏。一旦心脏停止（asystolic），消费税和从胸部和地点取出心脏在寒冷的Krebs-Henseleit缓冲液的浴（〜3-8°C）。 </li></ol></li><li>复活。 <ol><li>导管插入大血管和利用可见心的方法准备心脏（3）。一旦灌注心脏达到37±5℃，通过经由心外膜贴片电极递送34Ĵ冲击到心室恢复天然窦性心律。 </li></ol></li></ol> 5. 在体外研究范式<ol><li>连续监测60分钟后复苏的心脏功能。连续监测在2D模式下超声心动图与短轴切面:刚二尖瓣低于LV视图。 </li><li>计算心室壁厚度和射血分数每10分钟后复苏（见下文）。在评估期间采取从-5分钟冠状窦（1毫升），并每5分钟样本。 </li><li>监控。 <ol><li>监视左和右心室，包括压力，流量和量的变化两者血液动力学。 </li></ol></li><li>超声心动图:射血分数。 <ol><li>直接放在心脏经胸回声探测，捕获中期乳头短轴观。用公式计算射血分数（EF％）的4室的工作模式，每10分钟:LV舒张末期容积 - 左心室收缩末期容积×100 / LV舒张末期容积</li></ol></li><li>超声心动图:左室壁厚度。 <ol><li>以10分钟的间隔一个小时后复苏测量从一个中期的乳头短轴窗口室壁厚度（厘米）。测量使用屏幕卡尺左心室的四角和取平均。 </li></ol></li><li>心肌代谢。 <ol><li>放置steeraBLE导管冠状窦（CS）。从样品前复活的CS 5分钟，每5分钟后复苏1小时1毫升。测试乳酸盐和葡萄糖水平的样品;其他血液气体也可以被评估。 </li></ol></li></ol>

<ol>
	<li>Richardson, E. S., Rolfes, C., Woo, S. O., Elmquist, W. F., Benditt, D. G., Iaizzo, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+Responses+to+the+Intrapericardial+Delivery+of+Metoprolol:+Target+Delivery+Compared+to+Intravenous+Administration.">Cardiac Responses to the Intrapericardial Delivery of Metoprolol: Target Delivery Compared to Intravenous Administration.</a> J. Cardiovasc. Translational Research. 5 (4), 535-540 (2012).</li><li>Xiao, Y. F., Sigg, D. C., Ujhelyi, M. R., Wilhelm, J. J., Richardson, E. S., Iaizzo, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pericardial+delivery+of+omega-3+fatty+acid:+a+novel+approach+to+reducing+myocardial+infarct+sizes+and+arrhythmias.">Pericardial delivery of omega-3 fatty acid: a novel approach to reducing myocardial infarct sizes and arrhythmias.</a> Am J Physiol Heart Circ Physiol. 294 (5), 2212-2218 (2008).</li><li>Chinchoy, 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=Isolated+four-chamber+working+swine+heart+model.">Isolated four-chamber working swine heart model.</a> Ann Thorac Surg. 70 (5), 1607-1614 (2000).</li><li>Moreno, R., Waxman, S., Rowe, K., Verrier, R. 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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrapericardial+ibutilide+administration+fails+to+terminate+pacing-induced+sustained+atrial+fibrillation+in+dogs.">Intrapericardial ibutilide administration fails to terminate pacing-induced sustained atrial fibrillation in dogs.</a> Cardiovas Drugs Ther. 18 (4), 269-277 (2004).</li><li>Kumar, K., Nguyen, K., Waxman, S., Nearing, B. D., Wellenius, G. A., Zhao, S. X., Verrier, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potent+antifibrillatory+effects+of+intrapericardial+nitroglycerin+in+the+ischemic+porcine+heart.">Potent antifibrillatory effects of intrapericardial nitroglycerin in the ischemic porcine heart.</a> J Am Coll Cardiol. 41 (10), 1831-1837 (2003).</li><li>Fei, L., Baron, A. D., Henry, D. P., Zipes, D. 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The authors have nothing to disclose

这里，我们已经证明用于测试目标递送化合物的潜在功效进入心包空间中的独特的方法。本研究范例可以用于测试目前市场释放药物产品或实验化合物，从而提供了直接的平移应用到各种临床设置。在这一治疗策略有交付药品，心包内的两个主要的临床应用:1）开放式心脏或微创心脏外科手术; 2）心脏移植和器官保存。此外，本研究范例具有可以被分析，以便更好地了解自己这些描述的治疗策略?...

目前在开放式心脏手术，医生使用抗心律失常等治疗药物全身。然而，这可能是许多患者，特别是那些谁已经在临床上损害问题。例如，静脉内治疗可导致血压或肾功能不全的全身滴;进一步，它们可以创建麻醉管理问题和/或其它长期副作用。 在这里，我们创建了一个模型来测试药物给药制剂进入心包空间的功效。例如，这种方法可用于测试抗心律失常药物，研究可能增加心脏功能和/或外科手术后的心肌的促进恢复化合物。已经有目标交付的治疗进入心包腔与静脉给药观察的优点: 例如 ，我们的实验室证明antiar的本地化交付节奏的药物，如美托洛尔，是针对心律失常的发生保护，同时尽量减少在血压1降低。这一目标的投放策略也提供了更高的管理焦点浓度，同时尽量减少系统水平的机会。例如，高含量的脂肪酸静脉内递送的浓度可能会导致溶血，但是心包输送最小化这一问题2。 本研究范例包括三个主要的目标，以确定心包递送化合物的功效:1） 在原位测定心房心室节点的不应期，心房和心室，治疗前和30分钟和60分钟后处理的; 2）相对治疗 30和60分钟后处理（经常被添加额外的时间点），它已被重新加进3 INC后的心脏3）功能分析之前， 现场 AF负担泸定血流动力学监测，心脏率，从冠状静脉窦采样心脏代谢（乳酸和葡萄糖），射血分数（EF％）和室壁厚度（厘米）来监控每10分钟后复苏这一治疗策略模型使我们能够观察到的影响药剂可以减少期间和心脏手术或移植后心律失常和/或缺血性损伤的发生率。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>SelectSecure&#174; 3830 lead</td>
			<td>Medtronic</td>
			<td>N/A</td>
			<td>Pacing Lead&#160;</td>
		</tr>
		<tr>
			<td>C304 Deflectable Catheter&#160;</td>
			<td>Medtronic</td>
			<td>N/A</td>
			<td>Steerable catheter for placing leads&#160;</td>
		</tr>
		<tr>
			<td>SelectSecure&#174; 3830 lead</td>
			<td>Medtronic</td>
			<td>N/A</td>
			<td>active fixation pacing leads&#160;</td>
		</tr>
		<tr>
			<td>Grass S48 Stimulator</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Electrical Stimulator&#160;</td>
		</tr>
		<tr>
			<td>Premium 6500 Unipolar Pacing&#160;</td>
			<td></td>
			<td>N/A</td>
			<td>Plunge pacing lead for LAA</td>
		</tr>
		<tr>
			<td>EnSite&#8482; Cardiac Mapping</td>
			<td></td>
			<td>N/A</td>
			<td>Electrophysiology mapping system</td>
		</tr>
		<tr>
			<td>CareLink Programer 2092</td>
			<td>Medtronic</td>
			<td>N/A</td>
			<td>programmer for pacing leads</td>
		</tr>
		<tr>
			<td>GEM II &#174; pacemaker&#160;</td>
			<td>Medtronic</td>
			<td>N/A</td>
			<td>pacemaker can&#160;</td>
		</tr>
		<tr>
			<td>DLP &#174; Aortic Root Cannula</td>
			<td>Medtronic</td>
			<td>N/A</td>
			<td>aortic root cannula for transplant&#160;</td>
		</tr>
		<tr>
			<td>C-Arm Fluoroscopy</td>
			<td>Ziehm&#160;</td>
			<td>N/A</td>
			<td>fluoroscopic imaging&#160;</td>
		</tr>
		<tr>
			<td>Oscilliscope</td>
			<td>Tektronix&#160;</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>11F Hemostasis introducer</td>
			<td>SafeSheath</td>
			<td>N/A</td>
			<td>Hemostasis introducers</td>
		</tr>
		<tr>
			<td>Swan-Ganz Catheter 8.0F</td>
			<td>ICU Medical&#160;</td>
			<td>N/A</td>
			<td>thermal dilution catheter&#160;</td>
		</tr>
		<tr>
			<td>Venogram balloon&#160;</td>
			<td>Oscor</td>
			<td>N/A</td>
			<td>pressure monitoring&#160;</td>
		</tr>
		<tr>
			<td>Ultraview SL</td>
			<td>Spacelabs</td>
			<td>N/A</td>
			<td>EKG and blood pressure&#160;</td>
		</tr>
		<tr>
			<td>s/5 Avance</td>
			<td>General Electric</td>
			<td>N/A</td>
			<td>Anesthesia machine</td>
		</tr>
		<tr>
			<td>Atrial 6492 &#8211; Unipolar Temporary Atrial Pacing Lead</td>
			<td>Medtronic</td>
			<td>N/A</td>
			<td>temporary pacing lead&#160;</td>
		</tr>
		<tr>
			<td>VIVID i</td>
			<td>General Electric</td>
			<td></td>
			<td>2D electrocardiography unit&#160;</td>
		</tr>
	</tbody>
</table>

testing the efficacy of pharmacological agents in a pericardial target delivery model in the swine

迄今为止，许多药剂用于治疗或预防心脏直视例心律失常创建不希望的全身性副作用。例如，静脉内给予抗心律失常药物可以产生全身性的压降在已经受损的心脏的病人。在执行开放式心脏手术，外科医生通常要么创建一个小口或形成心包支架创造适宜的操作领域。此为目标的药理递送（抗心律不齐或缺血预处理剂）直接连接到心肌组织而不不良副作用访问收益的机会。 我们已经制定了心包液中测试药物制剂为目标交付猪模型。而完全麻醉，每只动物用Swan-Ganz导管仪器以及左和右心室压力导管和起搏引线置于右心耳和右心室。然后进行内侧胸骨和心包访问摇篮创建的;暴跌起搏导线被放置在左心耳和双极起搏引线放置在左心室。利用程序员和心脏标测系统，房室结（AVN）的不应期，心房和心室测定。此外，制作心房颤动（AF）利用诱导房颤一个草刺激和时间进行了观察。这些测量被处理，以及30分钟和心包治疗后60分钟之前进行。对选定的研究中加入额外的时间点。随后心脏被cardiopleged并在四室的工作模式重新加进的。压力测量和功能进行了记录复苏后1小时。此治疗策略模型使我们能够观察药理学试剂的可能降低期间​​和AF心律失常和/或缺血性损伤，发生率的影响之三心脏直视手术。

这些结果是可利用在猪的药理学试剂的这个目标传递模型要收集的数据的特性。此数据显示在下面的一个DHA输液原位这些心室有效不应期（VERP）显着增加。此外，该数据确立了较体外控制的DHA左心室压力的增加。相比于在几个时间点控制在DHA处理的心脏的左心室压力均显著高。该数据证实了原位和体外试验治疗策略药理窗口。 <p class="jove_content" fo:keep-together.within-page...

Watch this Scientific Journal Video about Testing the Efficacy of Pharmacological Agents in a Pericardial Target Delivery Model in the Swine at JoVE.com

Testing the Efficacy of Pharmacological Agents in a Pericardial Target Delivery Model in the Swine

We have developed a swine model for the target delivery of pharmacological agents within the pericardial space/fluid. Using this approach, the relative benefits of administered agents on induced atrial fibrillation, relative refractory periods and/or ischemic protection can be investigated.

药理试验药物的疗效在心包目标交付模型在猪

To date, many pharmacological agents used to treat or prevent arrhythmias in open-heart cases create undesired systemic side effects. For example, ...

testing-efficacy-pharmacological-agents-pericardial-target-delivery

Research

Education

JoVE Core

JoVE Journal

Pharmacology

Medicine

Unit 1

Cardiovascular Drugs: Antiarrhythmic and Heart Failure Drugs

SCIENTISTS IN ACTION

8.2K 次观看.  University of Minnesota. We have developed a swine model for the target delivery of pharmacological agents within the pericardial space/fluid. Using this approach, the relative benefits of administered agents on induced atrial fibrillation, relative refractory periods and/or ischemic protection can be investigated. 

视频: Testing the Efficacy of Pharmacological Agents in a Pericardial Target Delivery Model in the Swine

Making Sense of Listening: The IMAP Test Battery

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the abilities to extract and use the information encoded in the auditory signal. We refer to these as listening skills (or auditory processing AP). Deficits in these skills are associated with delayed language and literacy development, though the nature of the relevant deficits and their causal connection with these delays is hotly debated.
When a child is referred to a health professional with normal hearing and unexplained difficulties in listening, or associated delays in language or literacy development, they should ideally be assessed with a combination of psychoacoustic (AP) tests, suitable for children and for use in a clinic, together with cognitive tests to measure attention, working memory, IQ, and language skills. Such a detailed examination needs to be relatively short and within the technical capability of any suitably qualified professional. Current tests for the presence of AP deficits tend to be poorly constructed and inadequately validated within the normal population. They have little or no reference to the presenting symptoms of the child, and typically include a linguistic component. Poor performance may thus reflect problems with language rather than with AP. To assist in the assessment of children with listening difficulties, pediatric audiologists need a single, standardized child-appropriate test battery based on the use of language-free stimuli.
We present the IMAP test battery which was developed at the MRC Institute of Hearing Research to supplement tests currently used to investigate cases of suspected AP deficits. IMAP assesses a range of relevant auditory and cognitive skills and takes about one hour to complete. It has been standardized in 1500 normally-hearing children from across the UK, aged 6-11 years. Since its development, it has been successfully used in a number of large scale studies both in the UK and the USA. IMAP provides measures for separating out sensory from cognitive contributions to hearing. It further limits confounds due to procedural effects by presenting tests in a child-friendly game-format. Stimulus-generation, management of test protocols and control of test presentation is mediated by the IHR-STAR software platform. This provides a standardized methodology for a range of applications and ensures replicable procedures across testers. IHR-STAR provides a flexible, user-programmable environment that currently has additional applications for hearing screening, mapping cochlear implant electrodes, and academic research or teaching.

A test battery (IMAP) for performing an in-depth assessment of auditory and cognitive abilities contributing to listening skills is described. It is quick to administer, child-friendly and free from linguistic confounds. Stimulus generation and protocol management are controlled via a software platform (IHR-STAR) to ensure replicable procedures.

听力:IMAP测试电池的决策意识

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the ...

科研

医学

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1 and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo mutations. This is the case for autism and schizophrenia3. The male-to-female ratio of mutation rate is estimated at about 4&#x2013;6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo mutations would more frequently come from males, particularly older males4. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.

Molecular genetic strategy for finding de novo mutations causing common disorders such as autism and schizophrenia.

一项战略，以确定在常见的疾病，如自闭症和精神分裂症的新突变

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, ...

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, ...

Clinical Testing and Spinal Cord Removal in a Mouse Model for Amyotrophic Lateral Sclerosis (ALS)

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder resulting in progressive degeneration of motoneurons. Peak of onset is around 60 years for the sporadic disease and around 50 years for the familial disease. Due to its progressive course, 50% of the patients die within 30 months of symptom onset. In order to evaluate novel treatment options for this disease, genetic mouse models of ALS have been generated based on human familial mutations in the SOD gene, such as the SOD1 (G93A) mutation. Most important aspects that have to be evaluated in the model are overall survival, clinical course and motor function. Here, we demonstrate the clinical evaluation, show the conduction of two behavioural motor tests and provide quantitative scoring systems for all parameters. Because an in depth analysis of the ALS mouse model usually requires an immunohistochemical examination of the spinal cord, we demonstrate its preparation in detail applying the dorsal laminectomy method. Exemplary histological findings are demonstrated. The comprehensive application of the depicted examination methods in studies on the mouse model of ALS will enable the researcher to reliably test future therapeutic options which can provide a basis for later human clinical trials.

Clinical Testing and Spinal Cord Removal in a Mouse Model for Amyotrophic Lateral Sclerosis ALS

A mouse model for amyotrophic lateral sclerosis (ALS) is examined clinically and behaviorally. As a prerequisite for an accompanying immunohistological analysis the preparation of the spinal cord is depicted in detail.

临床试验和脊髓去除在肌萎缩性侧索硬化症的小鼠模型（ALS）

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder resulting in progressive degeneration of motoneurons. Peak of onset is around 60 ...

Slow-release Drug Delivery through Elvax 40W to the Rat Retina: Implications for the Treatment of Chronic Conditions

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these diseases. Chronic retinal diseases such as retinal oedema or neovascularization usually require multiple intraocular injections to effectively treat the condition. However, the risks associated with these injections increase with repeated delivery of the drug. Therefore, alternative delivery methods need to be established in order to minimize the risks of reinjection. Several other investigations have developed methods to deliver drugs over extended time, through materials capable of releasing chemicals slowly into the eye. In this investigation, we outline the use of Elvax 40W, a copolymer resin, to act as a vehicle for drug delivery to the adult rat retina. The resin is made and loaded with the drug. The drug-resin complex is then implanted into the vitreous cavity, where it will slowly release the drug over time. This method was tested using 2-amino-4-phosphonobutyrate (APB), a glutamate analogue that blocks the light response of the retina. It was demonstrated that the APB was slowly released from the resin, and was able to block the retinal response by 7 days after implantation. This indicates that slow-release drug delivery using this copolymer resin is effective for treating the retina, and could be used therapeutically with further testing.

This paper details how Elvax 40W can be used as a slow-release method for drug delivery to the adult rat retina. The protocol for preparing, loading, and delivering the drug-resin complex to the eye is described.

通过40W的Elvax缓释给药的大鼠视网膜:对慢性疾病的治疗

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these ...

Surgical Placement of Catheters for Long-term Cardiovascular Exercise Testing in Swine

This protocol describes the surgical procedure to chronically instrument swine and the procedure to exercise swine on a motor-driven treadmill. Early cardiopulmonary dysfunction is difficult to diagnose, particularly in animal models, as cardiopulmonary function is often measured invasively, requiring anesthesia. As many anesthetic agents are cardiodepressive, subtle changes in cardiovascular function may be masked. In contrast, chronic instrumentation allows for measurement of cardiopulmonary function in the awake state, so that measurements can be obtained under quiet resting conditions, without the effects of anesthesia and acute surgical trauma. Furthermore, when animals are properly trained, measurements can also be obtained during graded treadmill exercise.
Flow probes are placed around the aorta or pulmonary artery for measurement of cardiac output and around the left anterior descending coronary artery for measurement of coronary blood flow. Fluid-filled catheters are implanted in the aorta, pulmonary artery, left atrium, left ventricle and right ventricle for pressure measurement and blood sampling. In addition, a 20 G&#160;catheter is positioned in the anterior interventricular vein to allow coronary venous blood sampling.
After a week of recovery, swine are placed on a motor-driven treadmill, the catheters are connected to pressure and flow meters, and swine are subjected to a five-stage progressive exercise protocol, with each stage lasting 3 min. Hemodynamic signals are continuously recorded and blood samples are taken during the last 30 sec of each exercise stage.
The major advantage of studying chronically instrumented animals is that it allows serial assessment of cardiopulmonary function, not only at rest but also during physical stress such as exercise. Moreover, cardiopulmonary function can be assessed repeatedly during disease development and during chronic treatment, thereby increasing statistical power and hence limiting the number of animals required for a study.

Here we present a protocol to assess cardiopulmonary function in awake swine, at rest and during graded treadmill exercise. Chronic instrumentation allows for repeated hemodynamic measurements uninfluenced by cardiodepressive anesthetic agents.

导管手术放置了长期的心血管运动试验猪

This protocol describes the surgical procedure to chronically instrument swine and the procedure to exercise swine on a motor-driven treadmill. Early ...

Standardized Model of Ventricular Fibrillation and Advanced Cardiac Life Support in Swine

Cardiopulmonary resuscitation after cardiac arrest, independent of its origin, is a regularly encountered medical emergency in hospitals as well as preclinical settings. Prospective randomized trials in human subjects are difficult to design and ethically ambiguous, which results in a lack of evidence-based therapies. The model presented in this report represents one of the most common causes of cardiac arrests, ventricular fibrillation, in a standardized setting in a large animal model. This allows for reproducible observations and various therapeutic interventions under clinically accurate conditions, hence facilitating the generation of better evidence and eventually the potential for improved medical treatment.

Cardiopulmonary resuscitation and defibrillation are the only effective therapeutic options during cardiac arrest caused by ventricular fibrillation. This model presents a standardized regimen to induce, assess, and treat this physiological state in a porcine model, thus providing a clinical approach with various opportunities for data collection and analysis.

猪心室颤动和高级心脏生命支持标准化模型

Cardiopulmonary resuscitation after cardiac arrest, independent of its origin, is a regularly encountered medical emergency in hospitals as well as ...

Intratracheal Administration of Dry Powder Formulation in Mice

In the development of inhalable dry powder formulations, it is essential to evaluate their biological activities in preclinical animal models. This paper introduces a noninvasive method of intratracheal delivery of dry powder formulation in mice. A dry powder loading device that consists of a 200 &#181;L gel loading pipette tip connected to an 1 mL syringe via a three-way stopcock is presented. A small amount of dry powder (1-2 mg) is loaded into the pipette tip and dispersed by 0.6 mL of air in the syringe. Because pipette tips are disposable and inexpensive, different dry powder formulations can be loaded into different tips in advance. Various formulations can be evaluated in the same animal experiment without device cleaning and dose refilling, thereby saving time and eliminating the risk of cross-contamination from residual powder. The extent of powder dispersion can be inspected by the amount of powder remaining in the pipette tip. A protocol of intubation in mouse with a custom-made light source and a guiding cannula is included. Proper intubation is one of the key factors that influences the intratracheal delivery of dry powder formulation to the deep lung region of the mouse.

Dry powder formulations for inhalation have great potential in treating respiratory diseases. Before entering human studies, it is necessary to evaluate the efficacy of the dry powder formulation in preclinical studies. A simple and noninvasive method of the administration of dry powder in mice through the intratracheal route is presented.

小鼠干粉配方的内经管理

In the development of inhalable dry powder formulations, it is essential to evaluate their biological activities in preclinical animal models. This paper ...

Large Animal Model for Evaluating the Efficacy of the Gene Therapy in Ischemic Heart

Coronary artery disease is one of the significant causes of mortality and morbidity worldwide. Despite the progression of current therapeutics, a considerable proportion of coronary artery disease patients remain symptomatic. Gene therapy-mediated therapeutic angiogenesis offers a novel therapeutic method for improving myocardial perfusion and relieving symptoms. Gene therapy with different angiogenic factors has been studied in few clinical trials. Due to the novelty of the method, the progress of myocardial gene therapy is a continuous path from bench to bedside. Therefore, large animal models are needed for evaluating the safety and efficacy. The more the large animal model identifies the original disease and the endpoints used in clinics, the more predictable outcomes are from clinical trials. Here, we introduce a large animal model for evaluating the efficacy of the gene therapy in the ischemic porcine heart. We use clinically relevant imaging methods such as ultrasound imaging and 15H2O-PET. For targeting the gene transfers into the desired area, electroanatomical mapping is used. The aim of this method is: (1) to mimic chronic coronary artery disease, (2) to induce therapeutic angiogenesis at hypoxic areas of the heart, and (3) to evaluate the safety and efficacy of the gene therapy by using relevant endpoints.

Myocardial gene therapy for ischemic heart disease holds great promise for future therapeutics. Here, we introduce a large animal model for evaluating the efficacy of gene therapy in the ischemic heart.

用于评估基因治疗缺血性心脏疗效的大型动物模型

Coronary artery disease is one of the significant causes of mortality and morbidity worldwide. Despite the progression of current therapeutics, a ...

Endotracheal Intubation Using a Flexible Intubation Endoscope as a Standardized Model for Safe Airway Management in Swine

Endotracheal intubation is often a basic requirement for translational research in porcine models for various interventions that require a secured airway or high ventilation pressures. Endotracheal intubation is a challenging skill,&#160;requiring a minimum number of successful endotracheal intubations to achieve a high success rate under optimal conditions, which is often unachievable for non-anaesthesiology researchers. Due to the specific porcine airway anatomy, a difficult airway can usually be assumed. The impossibility of establishing a secure airway can result in injury, adverse events, or death of the laboratory animal. Using a prospective, randomized, controlled evaluation approach, it has been shown that fiberoptic-assisted endotracheal intubation takes longer but has a higher first-pass success rate than conventional intubation without causing clinically relevant drops in oxygen saturation. This model presents a standardized regimen for endoscopically guided endotracheal intubation, providing a secured airway, especially for researchers who are inexperienced in the technique of endotracheal intubation via direct laryngoscopy. This procedure is expected to minimize animal suffering and unnecessary animal losses.

The use of pigs in research has increased in recent years. Nevertheless, pigs are characterized by difficult airway anatomy. By demonstrating how to perform endoscopically guided endotracheal intubation, the present protocol aims to further increase laboratory animals' safety to avoid animal suffering and unnecessary death.

使用柔性插管内窥镜作为猪安全气道管理的标准化模型的气管插管

Endotracheal intubation is often a basic requirement for translational research in porcine models for various interventions that require a secured airway ...