Name: an intact pericardium ischemic rodent model
Uploaded: 2021-09-02T14:00:00
Description: Watch this Scientific Journal Video about An Intact Pericardium Ischemic Rodent Model at JoVE.com

8-14周龄的雄性和雌性C57BL / 6J小鼠用于这些实验。该协议已获得卡尔加里大学动物护理委员会的伦理批准，并遵循所有动物护理指南。
1. 小鼠准备和手术
<ol><li>对手术工具进行消毒（通过珠状消毒器或高压灭菌器）。</li>
	<li>称量小鼠术前体重和镇痛剂量。</li>
	<li>将小鼠置于具有4%异氟醚和800mL / min氧气的诱导盒中。通过捏住脚趾并观察动物缺乏反射来确认麻醉平面。</li>
	<...

<ol>
	<li>Virani, S. S., 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=Heart+disease+and+stroke+statistics-2020+update:+A+report+from+the+American+Heart+Association.">Heart disease and stroke statistics-2020 update: A report from the American Heart Association.</a> Circulation. 141, 139 (2020).</li><li>Iismaa, 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=Comparative+regenerative+mechanisms+across+different+mammalian+tissues.">Comparative regenerative mechanisms across different mammalian tissues.</a> NPJ Regenerative Medicine. 3 (6), (2018).</li><li>Bayat, 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=Progressive+heart+failure+after+myocardial+infarction+in+mice.">Progressive heart failure after myocardial infarction in mice.</a> Basic Research in Cardiology. 97 (3), 206-213 (2002).</li><li>Virag, J. A., Lust, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronary+artery+ligation+and+intramyocardial+injection+in+a+murine+model+of+infarction.">Coronary artery ligation and intramyocardial injection in a murine model of infarction.</a> Journal of Visualized Experiments. 52, 2581 (2011).</li><li>De Villiers, C., Riley, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+myocardial+infarction:+comparing+permanent+ligation+and+ischaemia-reperfusion.">Mouse models of myocardial infarction: comparing permanent ligation and ischaemia-reperfusion.</a> Disease Models &amp; Mechanisms. 13 (11), (2020).</li><li>Borlaug, B. A., Reddy, Y. N. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+pericardium+in+heart+failure:+Implications+for+pathophysiology+and+treatment.">The role of the pericardium in heart failure: Implications for pathophysiology and treatment.</a> JACC Heart Failure. 7 (7), 574-585 (2019).</li><li>Pfaller, M. R., 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=The+importance+of+the+pericardium+for+cardiac+biomechanics:+from+physiology+to+computational+modeling.">The importance of the pericardium for cardiac biomechanics: from physiology to computational modeling.</a> Biomechanics and Modeling in Mechanobiology. 18 (2), 503-529 (2019).</li><li>Deniset, J. F., 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=Gata6(+)+Pericardial+Cavity+Macrophages+Relocate+to+the+Injured+Heart+and+Prevent+Cardiac+Fibrosis.">Gata6(+) Pericardial Cavity Macrophages Relocate to the Injured Heart and Prevent Cardiac Fibrosis.</a> Immunity. 51 (1), 131-140 (2019).</li><li>Weber, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+targeting+of+the+pleural+space+by+intercostal+approach.">Immune targeting of the pleural space by intercostal approach.</a> BMC Pulmonary Medicine. 15, 14 (2015).</li><li>Nakatani, T., Shinohara, H., Fukuo, Y., Morisawa, S., Matsuda, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pericardium+of+rodents:+pores+connect+the+pericardial+and+pleural+cavities.">Pericardium of rodents: pores connect the pericardial and pleural cavities.</a> The Anatomical Record. 220, 132-137 (1988).</li><li>Tyberg, J. V., 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=The+relationship+between+pericardial+pressure+and+right+atrial+pressure:+an+intraoperative+study.">The relationship between pericardial pressure and right atrial pressure: an intraoperative study.</a> Circulation. 73, 428-432 (1986).</li><li>Hamilton, D. R., Sas, R., Semlacher, R. A., Kieser Prieur, T. M., Tyberg, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relationship+between+left+and+right+pericardial+pressures+in+humans:+an+intraoperative+study.">The relationship between left and right pericardial pressures in humans: an intraoperative study.</a> The Canadian Journal of Cardiology. 27, 346-350 (2011).</li><li>Park, D. S. 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=Human+pericardial+proteoglycan+4+(lubricin):+Implications+for+postcardiotomy+intrathoracic+adhesion+formation.">Human pericardial proteoglycan 4 (lubricin): Implications for postcardiotomy intrathoracic adhesion formation.</a> The Journal of Thoracic and Cardiovascular Surgery. 156 (4), 1598-1608 (2018).</li></ol>

在啮齿动物封闭的心包中诱导心肌梗死是独一无二的，并且可能具有潜在的重要应用。该手术在很大程度上依赖于外科医生对啮齿动物模型和啮齿动物心脏解剖学的熟悉程度。成功还取决于在三个关键步骤中给予的护理：肋间肌切口和肋骨回缩（步骤1.11-1.13），产生梗死（步骤1.17）和动物恢复（步骤1.22-1.24）。
开胸术必须认真进行，以避免刺穿或撕裂心包。该方案最关键的?...

时至今日，心血管疾病（CVD）仍被公认为全球主要死亡原因，导致巨大的经济负担和患者生活质量的降低1。冠状动脉疾病（CAD）是CVD的一种亚型，在心肌梗死（MI）的发展中起着至关重要的作用，心肌梗死是导致死亡的主要原因。根据定义，心肌梗死是由于长时间缺血和缺氧对心肌组织造成不可逆损伤所致。心肌组织缺乏再生能力，因此损伤是永久性的，并导致心肌被纤维化疤痕取代，纤维化疤痕最初可以起到保护作用，但最终会导致不良心脏重塑和最终的心力衰竭2。
尽管CAD患者的管理在过去几十年中得到了显着改善，但继发于缺血的慢性心力衰竭（CHF）影响着全世界的许多患者。为了预防和管理这种流行病，有必要更广泛地了解潜在的机制并开发新的治疗方法。此外，过去的研究结果强调了全身治疗的局限性以及开发精确替代方案的必要性。鉴于研究人类心肌梗死的分子后遗症受到进入梗死组织的能力的影响，概括与CVD相关的人心肌梗死和充血性心力衰竭的特征和发展的动物模型是必不可少的。
由于理想的动物模型在结构和功能特征上与人类疾病非常相似，因此疾病病因应指导其概念。在冠状动脉疾病中，它是冠状动脉的慢性动脉粥样硬化性狭窄或急性血栓性闭塞。已经开发并应用于各种实验动物的不同方法，以诱导冠状动脉狭窄或闭塞。这些策略大致可分为两组：（1）机械操作冠状动脉以诱发心肌梗死和（2）加速动脉粥样硬化以促进冠状动脉狭窄导致心肌梗死。第一种策略通常涉及结扎冠状动脉或在动脉内放置支架。第二种方法倾向于改变动物的饮食以包括高脂肪/胆固醇食物。后一种方法的一些局限性包括缺乏对冠状动脉闭塞的时间和部位的控制。
相比之下，在动物模型中手术诱导心肌梗死或缺血有几个优点，例如冠状动脉事件的位置、精确的时间和范围，从而获得更可重复的结果。最广泛使用的方法是手术结扎左前降冠状动脉（LAD）。这些模型概括了人类对急性缺血性损伤的反应，以及进展到CHF 3。LAD手术最初是在较大的动物身上开发的，随着技术的进步，对啮齿动物等小动物的LAD手术变得更加可行4。在建立这样的模型时，小鼠由于各种原因而受到青睐，包括它们的相对可用性，住房费用低以及遗传操作能力。
使用LAD闭塞的缺血性心脏病的当代手术模型要求研究人员打开心包以暂时或永久地结扎动脉5。这种策略导致心包空间的破坏，心包空间基本上起着机械和润滑功能，以确保正常的心脏功能。打开心包的另一个缺点是失去动物的天然心包液及其各种细胞和蛋白质成分6，7。作为回应，我们开发了一种在保持心包完整的同时诱导心肌梗死的方法。除了最大限度地减少这种稳态环境的扰动外，这种方法还允许在引起心肌梗死后标记和追踪特定细胞。此外，这种方法更好地代表了人类环境中的心肌缺血性损伤。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Steri-350 Bead Sterilizer</td>
			<td>Inotech</td>
			<td>NC9449759</td>
			<td></td>
		</tr>
		<tr>
			<td>10% Formalin</td>
			<td>Millipore Sigma</td>
			<td>HT501128-4L</td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#181;m Cell strainer</td>
			<td>VWR</td>
			<td>CA21008-949</td>
			<td>Falcon, 352340</td>
		</tr>
		<tr>
			<td>70 &#181;m Cell strainer</td>
			<td>VWR</td>
			<td>CA21008-952</td>
			<td>Falcon, 352350</td>
		</tr>
		<tr>
			<td>ACK Lysis Buffer</td>
			<td>Thermo Fisher</td>
			<td>A1049201</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Insyte-W Catheter Needle 24 G X 3/4&#34;</td>
			<td>CDMV Inc</td>
			<td>108778</td>
			<td></td>
		</tr>
		<tr>
			<td>Betadine (10% povidone-iodine topical solution)</td>
			<td>CDMV Inc</td>
			<td>104826</td>
			<td></td>
		</tr>
		<tr>
			<td>Blunt Forceps</td>
			<td>Fine Science Tools</td>
			<td>FST 11000-12</td>
			<td></td>
		</tr>
		<tr>
			<td>BNP Ophthalmic Ointment</td>
			<td>CDMV Inc</td>
			<td>17909</td>
			<td></td>
		</tr>
		<tr>
			<td>Castroviejo Needle Driver</td>
			<td>Fine Science Tools</td>
			<td>FST 12061-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5810R</td>
			<td>Eppendorf</td>
			<td>22625101</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase I</td>
			<td>Millipore Sigma</td>
			<td>SCR103</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase XI</td>
			<td>Millipore Sigma</td>
			<td>C7657</td>
			<td></td>
		</tr>
		<tr>
			<td>Covidien 5-0 Polysorb Suture - CV-11 taper needle</td>
			<td>Medtronic Canada</td>
			<td>GL-890</td>
			<td></td>
		</tr>
		<tr>
			<td>Covidien 5-0 Polysorb Suture - PC-13 cutting needle</td>
			<td>Medtronic Canada</td>
			<td>SL-1659</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved Blunt Forceps</td>
			<td>Fine Science Tools</td>
			<td>FST 11009-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Dako Mounting Medium</td>
			<td>Agilen</td>
			<td>CS70330-2</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Millipore Sigma</td>
			<td>11284932001</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol, 100%</td>
			<td>Millipore Sigma</td>
			<td>MFCD00003568</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethicon 8-0 Ethilon Suture - BV-130-4 taper needle</td>
			<td>Johnson &#38; Johnson Inc.</td>
			<td>2815G</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiber-Optic Light</td>
			<td>Nikon</td>
			<td>2208502</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Forceps</td>
			<td>Fine Science Tools</td>
			<td>FST 11150-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoresbrite&#174; YG Carboxylate Microspheres 1.00 &#181;m</td>
			<td>Polysciences, Inc.</td>
			<td>15702</td>
			<td></td>
		</tr>
		<tr>
			<td>Geiger Thermal Cautery Unit</td>
			<td>World Precision Instruments</td>
			<td>501293</td>
			<td>Model 150-ST</td>
		</tr>
		<tr>
			<td>Hyaluronidase</td>
			<td>Millipore Sigma</td>
			<td>H4272</td>
			<td></td>
		</tr>
		<tr>
			<td>Isofluorane Vaporizer</td>
			<td>Harvard Apparatus</td>
			<td>75-0951</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane USP, 250 mL</td>
			<td>CDMV Inc</td>
			<td>108737</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic Fixator Retraction System</td>
			<td>Fine Science Tools</td>
			<td>18200-20</td>
			<td></td>
		</tr>
		<tr>
			<td>MX550D- 40 MHz probe</td>
			<td>Fujifilm- Visual Sonics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Needle Driver</td>
			<td>Fine Science Tools</td>
			<td>FST 12002-12</td>
			<td></td>
		</tr>
		<tr>
			<td>PE-10 Tubing</td>
			<td>Braintree Scienctific, Inc.</td>
			<td>PE10 50 FT</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Fine Science Tools</td>
			<td>FST 14184-09</td>
			<td></td>
		</tr>
		<tr>
			<td>SMZ-1B Stereo Microscope</td>
			<td>Nikon</td>
			<td>SMZ1-PS</td>
			<td></td>
		</tr>
		<tr>
			<td>VentElite Small Animal Ventilator</td>
			<td>Harvard Apparatus</td>
			<td>55-7040</td>
			<td></td>
		</tr>
		<tr>
			<td>Vetergesic (10 mL, 0.3mg/mL buprenorphine))</td>
			<td>CDMV Inc</td>
			<td>124918</td>
			<td>controlled drug</td>
		</tr>
		<tr>
			<td>Vevo 2100 Software</td>
			<td>Fujifilm-Visual Sonics</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

an intact pericardium ischemic rodent model

该方案表明，心包及其内容物在缺血性啮齿动物模型中起着重要的抗纤维化作用（冠状动脉结扎诱导心肌损伤）。大多数临床前心肌梗死模型需要破坏心包完整性并失去稳态细胞环境。然而，最近我们开发了一种诱导心肌梗塞的方法，该方法最大限度地减少心包损伤并保留心脏的常驻免疫细胞群。已经观察到冠状动脉结扎后心包间隙完整的小鼠的心脏功能恢复得到改善。该方法为研究心肌梗死后心包间隙的炎症反应提供了机会。标记技术的进一步发展可以与该模型相结合，以了解心包免疫细胞在调节驱动心脏重塑（包括纤维化）的炎症机制方面的命运和功能。

这种改良的冠状动脉结扎模型已经过优化，可实现可重复性和动物存活。然而，由于心脏引起的严重损伤，一些预期的术中和术后死亡率与手术有关。男性的标准死亡率（~25-35%）通常高于女性（~10-15%）。
通过改良冠状动脉结扎术成功诱导心肌梗死应通过心脏功能参数和结构特征的变化来证明。对于功能，在心肌梗死后3-4周内，通过超声心动图评估的左心室（LV）射血分数等?...

Watch this Scientific Journal Video about An Intact Pericardium Ischemic Rodent Model at JoVE.com

An Intact Pericardium Ischemic Rodent Model

该协议概述了在保留心包及其内容物的同时诱导小鼠心肌梗死的步骤。

完整的心包缺血性啮齿动物模型

This protocol has shown that the pericardium and its contents play an essential anti-fibrotic role in the ischemic rodent model (coronary ligation to ...

这种方法将使我们能够评估心脏周围的心包腔及其内容物在心肌梗塞后心脏修复中的作用。与用于模拟心肌梗死的传统冠状动脉结扎方法相比，我们的方法保持完整的心包腔，允许确定内容物并跟踪其在心脏缺血性损伤后的作用。了解心包腔的贡献和心脏损伤反应将为针对心包改善心脏愈合的治疗策略提供重要的见解。从麻醉后开始，约束小鼠的爪子并将其放在手术台上。按照手稿中所述准备小鼠进行手术后，使用商用呼吸机用2%异氟醚和100%氧气作为载气对小鼠进行通气。然后设定每分钟110次呼吸，潮气量250微升，呼气末正压为4毫米汞柱。将鼠标在其右侧滚动 30%，以定位胸部的左侧以进行手术。然后在胸部皮肤上做一个两到三厘米的横向切口，以观察左侧的胸大肌。从中线向外切开一厘米的切口，用于切开大胸大胸和小胸肌。在整个出血血管烧灼过程中避免过度出血。可视化第三和第四肋骨之间的肋间肌肉。在左肋间肌上做一个两厘米的切口，将空气引入胸腔，让心脏和肺从切口上脱落。为了切开肋间而不出血，请暂时停止呼吸机并使用烧灼装置扩大开口。使用牵开器，缩回肋骨以暴露心脏。然后在体视显微镜下观察心包和下面的心脏。轻轻地将镊子放在心包表面，以减少心包和潜在的心脏运动。通过追踪LAD冠状动脉在左附件下的出现，在视觉上地标它。使用微针驱动器，引导适当的缝合线穿过LAD下方的心包。观察LAD和心包另一侧出现的缝合线。代表永久性结扎模型，绑合缝合线以限制通过冠状动脉的血流，使左心室前部的漂白可见。然后借助剪刀修剪多余的缝合线。术后 72 小时每 12 小时皮下注射一次维持性镇痛药。将异氟醚降低到1%后，关闭异氟烷，同时保持氧气通气。一旦动物出现呼吸迹象，从嘴里取出 23 号气管插管并将小鼠放在恢复笼中以监测呼吸恢复。让小鼠在笼子中恢复，将笼子的一部分放在加热垫上以提供外部热源。通过评估切口和动物不适来监测小鼠的健康状况，持续七天。如有必要，给予抗生素以在侵入性手术后成功恢复。用氧气将鼠标保持在全身麻醉下后，将鼠标仰卧在加热的载物台平台上，并将爪子连接到心电图导联线上。然后擦伤胸部。使用换能器探头和接触凝胶采集心电图图像，并使用适当的软件进行分析。无创心电图评估后，关闭异氟醚并将小鼠放回笼中。用10%福尔马林固定心脏至少24小时。使用直剃刀刀片穿过右心室、室间隔和左心室切割样本，确保切口穿过梗死区的中心。然后石蜡包埋组织样品。然后将切割切片放置在安装盒中，以进一步嵌入石蜡，随后切成五微米厚的切片。将组织切片放置在载玻片上后，使用甲苯和分级酒精洗涤液用去离子水脱蜡。然后补充水分。接下来，在室温下用苦味酸中用0.1%天狼星红染色切片两个小时。用0.5%乙酸清洗切片三分钟，然后用70%乙醇冲洗一分钟。在安装以进行显微镜评估之前，使用与乙醇浓度增加和分级的洗涤相反的洗涤顺序使切片脱水。通过检查心脏的功能参数和结构特征，评估改良冠状动脉结扎术成功诱导心肌梗死的情况。对于功能，在心肌梗死后三到四周内通过超声心动图评估左心室射血分数降低。组织学染色表明，功能变化伴有左心室壁的显着纤维化，这通过对梗死后四周心脏横截面的picrosirius红色染色纤维化进行定量进一步证实，心包或完整的心包冠状动脉结扎模型。将荧光珠标记方法与流式细胞术分析相结合，提供了一种跟踪驻留Gata6阳性心包巨噬细胞的方法。重要的是，在心脏或血液中几乎没有检测到标记。心肌梗死后，可以通过流式细胞术和成像跟踪细胞的重新定位。显示了荧光珠标记的心包骨髓细胞的定量，伴或不伴有心肌梗死。需要注意防止在手术过程中无意中破坏或撕裂薄心包。该方法可以与谱系追踪细胞或效应阻断相结合，这将允许跟踪和确定心包成分对梗死心脏愈合反应的影响。我们已经确定心包腔在受伤后保护心脏，这部分是由常驻心包巨噬细胞介导的。这种方法开辟了心血管疾病的新研究领域。

an-intact-pericardium-ischemic-rodent-model

Research

JoVE Journal

Medicine

Gene Transfer for Ischemic Heart Failure in a Preclinical Model

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine their efficacy and safety.
Gene therapy using viral vectors is one of the most promising approaches for treating cardiac diseases. Viral delivery of various different genes by changing the carrier gene has immeasurable therapeutic potential.
In this video, the full process of an animal model of heart failure creation followed by gene transfer is presented using a swine model. First, myocardial infarction is created by occluding the proximal left anterior descending coronary artery. Heart remodeling results in chronic heart failure. Unique to our model is a fairly large scar which truly reflects patients with severe heart failure who require aggressive therapy for positive outcomes. After myocardial infarct creation and development of scar tissue, an intracoronary injection of virus is demonstrated with simultaneous nitroglycerine infusion. Our injection method provides simple and efficient gene transfer with enhanced gene expression. This combination of a myocardial infarct swine model with intracoronary virus delivery has proven to be a consistent and reproducible methodology, which helps not only to test the effect of individual gene, but also compare the efficacy of many genes as therapeutic candidates.

A method of gene transfer for the treatment of ischemic heart failure is described using a swine model of myocardial infarction. Our simple and reproducible method enables us to readily evaluate the efficacy of various gene transfers with a very simple and reproducible way.

缺血性心力衰竭在临床前模型的转基因

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine ...

科研

医学

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

Muscle strains are one of the most common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and physical exam alone, however the clinical presentation can vary greatly depending on the extent of injury, the patient's pain tolerance, etc. In patients with muscle injury or muscle disease, assessment of muscle damage is typically limited to clinical signs, such as tenderness, strength, range of motion, and more recently, imaging studies. Biological markers, such as serum creatine kinase levels, are typically elevated with muscle injury, but their levels do not always correlate with the loss of force production. This is even true of histological findings from animals, which provide a "direct measure" of damage, but do not account for all the loss of function. Some have argued that the most comprehensive measure of the overall health of the muscle in contractile force. Because muscle injury is a random event that occurs under a variety of biomechanical conditions, it is difficult to study. Here, we describe an in vivo animal model to measure torque and to produce a reliable muscle injury. We also describe our model for measurement of force from an isolated muscle in situ. Furthermore, we describe our small animal MRI procedure.

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

An in vivo animal model of injury is described. The method takes advantage of the subcutaneous position of the fibular nerve. Velocity, timing of muscle activation, and arc of motion are all pre-determined and synchronized using commercial software. Post injury changes are monitored in vivo using MR imaging/spectroscopy.

一个在体内收缩引起的损伤，非侵入性的监测恢复啮齿动物模型

Muscle strains are one of the most  common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and  physical ...

A Simplified Technique for Producing an Ischemic Wound Model

One major obstacle in current diabetic wound research is a lack of an ischemic wound model that can be safely used in diabetic animals. Drugs that work well in non-ischemic wounds may not work in human diabetic wounds because vasculopathy is one major factor that hinders healing of these wounds. We published an article in 2007 describing a rabbit ear ischemic wound model created by a minimally invasive surgical technique. Since then, we have further simplified the procedure for easier operation. On one ear, three small skin incisions were made on the vascular pedicles, 1-2 cm from the ear base. The central artery was ligated and cut along with the nerve. The whole cranial bundle was cut and ligated, leaving only the caudal branch intact. A circumferential subcutaneous tunnel was made through the incisions, to cut subcutaneous tissues, muscles, nerves, and small vessels. The other ear was used as a non-ischemic control. Four wounds were made on the ventral side of each ear. This technique produces 4 ischemic wounds and 4 non-ischemic wounds in one animal for paired comparisons. After surgery, the ischemic ear was cool and cyanotic, and showed reduced movement and a lack of pulse in the ear artery. Skin temperature of the ischemic ear was 1-10 &deg;C lower than that on the normal ear and this difference was maintained for more than one month. Ear tissue high-energy phosphate contents were lower in the ischemic ear than the control ear. Wound healing times were longer in the ischemic ear than in the non-ischemic ear when the same treatment was used. The technique has now been used on more than 80 rabbits in which 23 were diabetic (diabetes time ranging from 2 weeks to 2 years). No single rabbit has developed any surgical complications such as bleeding, infection, or rupture in the skin incisions. The model has many advantages, such as little skin disruption, longer ischemic time, and higher success rate, when compared to many other models. It can be safely used in animals with reduced resistance, and can also be modified to meet different testing requirements.

We have developed a minimally invasive technique to create a rabbit ischemic ear wound model by dividing the central artery and nerve and the cranial neurovascular bundle. A subcutaneous tunnel then cuts all subcutaneous tissues. This procedure causes minimal skin disruption and can be safely used in diabetic animals.

一个生产缺血性损伤模型的简化技术

One major obstacle in current diabetic wound research is a lack of an ischemic wound model that can be safely used in diabetic animals. Drugs that work ...

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

采用实时定量PCR检测供体细胞植入一个具有竞争力的小鼠骨髓移植模型

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

Rat Model of Photochemically-Induced Posterior Ischemic Optic Neuropathy

Posterior Ischemic optic neuropathy (PION) is a sight-devastating disease in clinical practice. However, its pathogenesis and natural history have&#160;remained poorly understood. Recently, we developed a reliable, reproducible animal model of PION and tested the treatment effect of some neurotrophic factors in this model1. The purpose of this video is to demonstrate our photochemically induced model of posterior ischemic optic neuropathy, and to evaluate its effects with retrograde labeling of retinal ganglion cells. Following surgical exposure of the posterior optic nerve, a photosensitizing dye, erythrosin B, is intravenously injected and a laser beam is focused onto the optic nerve surface. Photochemical interaction of erythrosin B and the laser during irradiation damages the vascular endothelium, prompting microvascular occlusion mediated by platelet thrombosis and edematous compression. The resulting ischemic injury yields a gradual but pronounced retinal ganglion cell dieback, owing to a loss of axonal input - a remote, injury-induced and clinically relevant outcome. Thus, this model provides a novel platform to study the pathophysiologic course of PION,&#160;and can be further optimized for testing therapeutic approaches for optic neuropathies as well as other CNS ischemic diseases.

The goal of this protocol is to photochemically induce ischemic injury to the posterior optic nerve in rat. This model is critical to studies of the pathophysiology of posterior ischemic optic neuropathy, and therapeutic approaches for this and other optic neuropathies, as well as of other CNS ischemic diseases.

光化学诱导后部缺血性视神经病变的大鼠模型

Posterior Ischemic optic neuropathy (PION) is a sight-devastating disease in clinical practice. However, its pathogenesis and natural history ...

A Piglet Model of Neonatal Hypoxic-Ischemic Encephalopathy

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the world&#8217;s 2.9 million neonatal deaths. Animal models of HIE have contributed to the understanding of the pathophysiology in HIE, and have highlighted the dynamic process that occur in brain injury due to perinatal asphyxia. Thus, animal studies have suggested a time-window for post-insult treatment strategies. Hypothermia has been tested as a treatment for HIE in pdiglet models and subsequently proven effective in clinical trials. Variations of the model have been applied in the study of adjunctive neuroprotective methods and piglet studies of xenon and melatonin have led to clinical phase I and II trials1,2. The piglet HIE model is further used for neonatal resuscitation- and hemodynamic studies as well as in investigations of cerebral hypoxia on a cellular level. However, it is a technically challenging model and variations in the protocol may result in either too mild or too severe brain injury. In this article, we demonstrate the technical procedures necessary for establishing a stable piglet model of neonatal HIE. First, the newborn piglet (&#60; 24 hr old, median weight 1500 g) is anesthetized, intubated, and monitored in a setup comparable to that found in a neonatal intensive care unit. Global hypoxia-ischemia is induced by lowering the inspiratory oxygen fraction to achieve global hypoxia, ischemia through hypotension and a flat trace amplitude integrated EEG (aEEG) indicative of cerebral hypoxia. Survival is promoted by adjusting oxygenation according to the aEEG response and blood pressure. Brain injury is quantified by histopathology and magnetic resonance imaging after 72 hr.

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 ...

Demonstration of the Rat Ischemic Skin Wound Model

The propensity for chronic wounds in humans increases with ageing, disease conditions such as diabetes and impaired cardiovascular function, and unrelieved pressure due to immobility. Animal models have been developed that attempt to mimic these conditions for the purpose of furthering our understanding of the complexity of chronic wounds. The model described herein is a rat ischemic skin flap model that permits a prolonged reduction of blood flow resulting in wounds that become ischemic and resemble a chronic wound phenotype (reduced vascularization, increased inflammation and delayed wound closure). It consists of a bipedicled dorsal flap with 2 ischemic wounds placed centrally and 2 non-ischemic wounds lateral to the flap as controls. A novel addition to this ischemic skin flap model is the placement of a silicone sheet beneath the flap that functions as a barrier and a splint to prevent revascularization and reduce contraction as the wounds heal. Despite the debate of using rats for wound healing studies due to their quite distinct anatomic and physiologic differences compared to humans (i.e., the presence of a panniculus carnosus muscle, short life-span, increased number of hair follicles, and their ability to heal infected wounds) the modifications employed in this model make it a valuable alternative to previously developed ischemic skin flap models.

The rat, due to its size, availability, and rather docile behavior, has been utilized as a research model for many years. The goal of this protocol is to utilize the rat as an ischemic skin wound healing model to provide valuable insight into the pathophysiology of chronic wounds.

在大鼠缺血性皮肤创伤模型的演示

The propensity for chronic wounds in humans increases with ageing, disease conditions such as diabetes and impaired cardiovascular function, and ...

The Rodent Model of Nonarteritic Anterior Ischemic Optic Neuropathy (rNAION)

Nonarteritic anterior ischemic optic neuropathy (NAION) is a focal ischemic lesion of the optic nerve that affects 1/700 individuals throughout their lifetime. NAION results in optic nerve edema, selective loss of the retinal ganglion cell neurons (RGCs) and atrophy of the optic nerve. A rodent model of NAION that expresses most NAION features and sequelae has been developed, which is applicable to both rats and mice. This model utilizes a focal laser application of 532 nm wavelength to illuminate a photoactive dye, Rose Bengal (RB), to cause capillary damage and leakage at the targeted anterior optic nerve (the laminar region). After rNAION induction, there is an early optic nerve ischemia, optic nerve edema, and intraneural inflammation, followed by selective RGC and axonal loss. Since the optic nerve is a CNS white matter tract, the rNAION model is applicable to mechanistic studies of selective white matter ischemia, as well as neuroprotective analyses and short and long-term mechanisms of glial and neuronal response to ischemia.

The Rodent Model of Nonarteritic Anterior Ischemic Optic Neuropathy rNAION

The following report describes how to replicate the rodent model of nonarteritic anterior ischemic optic neuropathy (rNAION), using the appropriate dye, contact lens and laser parameters. We also reveal the appropriate steps for evaluating the rNAION lesion in vivo.

非动脉炎性前部缺血性视神经病变的啮齿动物模型（rNAION）

Nonarteritic anterior ischemic optic neuropathy (NAION) is a focal ischemic lesion of the optic nerve that affects 1/700 individuals throughout their ...

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 ...

Oxygen-Induced Retinopathy Model for Ischemic Retinal Diseases in Rodents

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR model induction and its readouts in both mice and rats. Retinal neovascularization is induced in OIR by exposing rodent pups either to hyperoxia (mice) or alternating levels of hyperoxia and hypoxia (rats). The primary readouts of these models are the size of neovascular (NV) and avascular (AVA) areas in the retina. This preclinical in vivo model can be used to evaluate the efficacy of potential anti-angiogenic drugs or to address the role of specific genes in the retinal angiogenesis by using genetically manipulated animals. The model has some strain and vendor specific variation in the OIR induction which should be taken into consideration when designing the experiments.

Oxygen-induced retinopathy (OIR) can be used to model ischemic retinal diseases such as retinopathy of prematurity and proliferative diabetic retinopathy and to serve as a model for proof-of-concept studies in evaluating antiangiogenic drugs for neovascular diseases. OIR induces robust and reproducible neovascularization in the retina that can be quantified.

罗登斯缺血性视网膜疾病的氧诱导视网膜病变模型

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR ...