对塔基博士和 m. Funamoto 博士的技术支持给予了赞赏。

在实验之前, 所有的老鼠都应该得到一个星期的适应。所有动物的外科手术都应按照《动物护理和使用指南》 (www.nap.edu/catalog/5140.html) 或其他适当的道德准则进行。议定书应由动物福利委员会在适当的机构批准, 然后再继续。所有后续的程序必须在无菌条件下执行。
1. 准备体外循环电路
注: 佩戴个人防护用品, 包括手套、眼镜、清洁大衣或一次性长袍。
<ol><li>体外循环电路的设置<ol>
<li>将聚氯乙烯管与静脉储层连接, 预先设计为体外循环电路, 以及改良的新生儿膜肺, 如图 1所示。确保所有连接紧密, 不要漏水。</li>
		<li>根据制造商的协议, 将体外循环电路设置到滚筒泵装置。</li>
		<li>将滚筒泵保持在高度可调节的工作台上, 并将工作台的高度调整到实验台下面的10厘米处。</li>
	</ol>
</li>
	<li>体外循环电路的启动<ol>
<li>将12毫升羟乙基淀粉溶液与0.1 毫升肝素和0.5 毫升7% 碳酸氢钠溶液混合在一起启动体外循环电路。</li>
		<li>以11毫升的启动溶液为主要电路, 用泵辊轻轻旋转。把18口径的排气针放进储气库中。</li>
		<li>打膜肺几次到 deair, 与倾斜的肺。空气必须完全遮蔽, 以避免空气栓塞和氧合不足。在电路启动过程中, 通过设置在储层上的电热灯加热电路。</li>
	</ol>
</li>
</ol>2. 体外循环前的程序
注: 使用前应用70% 酒精或季铵盐化合物消毒手术现场和器械。
<ol><li>麻醉和动物设置<ol>
<li>麻醉一只在蒸发器中吸入3.0% 异氟醚混合空气的老鼠。把老鼠放在工作架上, 然后将16口径的套管插管到气管里。按照您当地的动物护理指南, 关于镇痛剂量和频率 (如: 丁丙诺啡0.005 毫克/千克) 注意: 老鼠必须在深度麻醉和失去反射。呼吸应该是有节奏的, 但不能被逮捕。</li>
		<li>将老鼠转到装有电热垫的手术台上。启动机械通气, 8 毫升/千克的潮汐量, 呼吸速率为70循环/分钟, 30% 的灵感氧分数监测的氧气传感器。</li>
		<li>用 1.5-2.0% 异氟醚维持麻醉, 并在体外循环启动时增加对氯胺酮/甲苯噻嗪的管理。</li>
		<li>用直肠探头监测直肠温度。通过调整热垫的温度并将电路置于散热灯上, 保持常温37摄氏度的体温。</li>
		<li>将大鼠仰卧位, 用针固定四肢。通过将心电图电极针设置为双侧肩和左腹, 对心率进行监测。放一条湿润的纱布或在眼睛上涂抹眼药膏以防止干燥。</li>
	</ol>
</li>
	<li>插管<ol>
<li>通过 sprayingby 喷雾70% 乙醇或另一种防腐溶液对全身表面进行消毒后, 在两侧腹股沟区和右颈部区域剃须剃刀毛。局部麻醉药 (如利多卡因) 应在进行皮肤切口前使用。注: 作为一种替代方法, 可使用切口部位的外科擦洗, 而不是全身喷雾70% 乙醇, 以避免体温下降。</li>
		<li>用剪刀切开皮肤 (大约5毫米) 在双侧腹股沟区域和右颈部区域, 并坦率地解剖组织以暴露右侧的主股动脉。从附近的静脉和神经仔细地分离动脉。结扎在股动脉末端由4-0 丝和紧张暴露。</li>
		<li>用微剪刀垂直向动脉切开右股动脉壁 (约1毫米), 并仔细 cannulate 24 口径静脉导管, 从切口到深度1厘米以监测全身动脉。动脉血中气体分压的压力和分析。</li>
		<li>从导管中管理肝素钠 (500 磅/千克)。</li>
		<li>按照步骤2.2.2 和 2.2.3 cannulate 24 口径静脉导管进入左股动脉, 作为体外循环回路的动脉输液线。</li>
		<li>将17口径的多孔 angiocatheter 插入右颈内静脉, 并将其推进右心房和下腔静脉。不要用力推导管, 因为容器很容易折断。将导管连接到体外循环回路进行静脉引流。</li>
		<li>用湿纱布覆盖每个空心区域以避免污染。</li>
	</ol>
</li>
</ol>3. 体外循环期间的程序
<ol><li>在体外循环期间, 将100% 氧气气体送到肺, 0.8 升/分, 将呼吸率降低到30周/分钟。动脉氧分压需要从200到 400 mmHg。</li>
	<li>在体外循环开始时, 将热垫的温度设置提高到最大值42摄氏度, 以减轻体外循环后体温的立即下降。当体温恢复到36摄氏度时, 将设定温度调整到37摄氏度。</li>
	<li>小心地启动体外循环流程, 密切关注水库中血液的体积。空的水库可能导致空气栓塞。如果储层中的血液体积减少, 通过调整工作台高度或改变引流导管的位置来降低泵流量。不要重新定位静脉引流导管, 这可能容易导致右心房穿孔和/或心律失常。</li>
	<li>增加并保持泵的流量为100毫升/千克/分钟, 而平均血压维持在 70 mmHg。当保持适当的血压时, 水库中至少有1毫升的小容积是可以接受的。如果水库中的血液少于1毫升, 则可能导致器官发生空气栓塞。</li>
	<li>如果血压不稳定, 增加2-3 毫升的启动方案的电路 (它可能导致贫血后, 体外循环)。</li>
</ol>4. 体外循环后的程序
<ol><li>钳住静脉引流管, 并将其从电路中取出。将血液中的剩余血逐渐注入动脉以维持血压。</li>
	<li>将呼吸率提高到70周/分钟。</li>
	<li>取出静脉引流导管和左动脉导管, 然后结扎近端和远端部位的血管。</li>
	<li>在体外循环结束后, 从右股动脉取出60分钟的动脉线。</li>
	<li>用生理盐水清洗伤口, 缝合伤口。</li>
	<li>检查动物的自发性呼吸, 结束麻醉, extubate 气管管。</li>
	<li>管理加热不育的等渗液, 并使用热垫和电热灯保持动物的温暖。经常检查动物的病情, 直到从麻醉中恢复。必要时提供呼吸支持。注意: 一旦动物开始走动, 热源应该从笼子的一部分移走, 让动物选择温暖或凉爽的一面。</li>
	<li>使动物远离其他动物的公司, 直到呼吸完全恢复。在完全恢复之前, 不要将动物退回公司。</li>
	<li>检查从麻醉恢复后的食物和水摄入量, 并提供适当的营养支持。管理止痛药, 检查是否有不适或疼痛的迹象。</li>
</ol>

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所有作者在商业支持方面没有什么可透露的。

在大鼠体外循环模型中, 体外循环后, 炎症细胞因子和 HMGB-1 的血清和肺表达水平是调节炎症反应的关键转录因子, 显著增加。以往的临床研究表明, 在心血管手术患者中, HMGB-1 水平的血清分泌增加了11, 体外循环期间血清 HMGB-1 水平与更严重的全身炎症反应综合征有关。体外循环后肺氧合功能障碍12。此外, 血清 HMGB-1 水平是一种独立的生物标志物, 用于预测重症肺炎和急性呼吸窘迫综合征 (ARDS) 患者重症监护病房的死亡率13。采用大鼠体外循环模型, 成功地模拟了严重的体外循环相关 ARDS 与炎症细胞因子和 HMGB-1 活化的关系。
大量研究与体外循环相关的系统性炎症已经进行了大鼠模型, 因为它们的作用, 以调查系统性炎症反应, 包括促炎细胞因子表达14, 核卡伯 B激活15, 通过黏附分子16和矩阵 metalloproteinase-9 活动17激活中性粒细胞。利用大鼠体外循环模型18, 对 HO-1 蛋白在体外循环期间活化的氧化应激也进行了研究。
近年来, 大鼠体外循环模型在各种程序中得到发展, 以模拟体外循环并发症的临床情况。心肌的酶学、遗传和组织学效应可以通过心跳骤停模型7进行研究。此外, 还报告了 DHCA 模型, 揭示了胸主动脉手术后不良脑结局的相关机制, 并探讨了潜在神经保护策略6。这些模型基于传统的大鼠体外循环模型, 对于评价体外循环的各种效果, 密切模仿心血管手术的临床情况, 具有重要价值。
该模型的生存率取决于调查人员的技术改进。与发病率有关的最关键因素是血管分离、心脏破裂和腹腔血肿, 通常发生于技术衰竭。良好的暴露, 适当的张力对血管, 温和插管是手术的要求, 成功。控制保留血液和附加溶液, 保持至少 60 mmHg 的平均血压, 对于避免极端贫血和下肢缺血有重要意义。空气栓塞到肠道和/或其他腹部器官也是至关重要的避免;一旦发生这种情况, 该过程就无法完成。
在这些手术的早期实践中, 由于插管难度较大, 体外循环后20% 的存活率达到 10%, 体外循环术后的生存期约为4小时。经过20种模型的经验, 在模型制备过程中血流动力学稳定, 存活率显著提高到近80%。
在建立该模型的初期, 体外循环电路总容积为3毫升, 高于目前的。因此, 在体外循环期间, 过量的胶体体积诱发严重贫血 (平均红细胞压积: 14.7%)19。通过降低体外循环电路体积和手术出血, 体外循环期间红细胞压积显著增加到 21.3%, 维持稳定的血流动力学。
该17口径多孔是非常适合从颈内静脉插管, 通过右心房静脉, 由于其适当的长度和柔软, 防止磨损的静脉壁。此外, 多孔和足够的内径允许足够的静脉回流和高成功率。由于其盲的方式, 将导管引入静脉内是技术上的困难, 容易穿孔静脉或心房壁。
动脉插管的大鼠股动脉倾向于导致肢体缺血与尾动脉或颈动脉20。在我们的经验中, 血流动力学的不稳定性 (平均 BP 小于 60 mmHg), 结扎股骨深动脉和严重贫血可能导致严重腿部损伤。维持血压, cannulating 股动脉的较高区域, 保持较高的红细胞压积, 是防止大鼠先天丰富的侧支灌注引起肢体缺血的有效方法。
避免儿茶酚胺和输血是必要的, 以准确地调查血液动力学, 生物化学和/或生理反应的检测材料和干预措施。在我们的模型过程中, 没有必要升压和输血。深层麻醉会增强炎症细胞因子的释放, 进而恶化器官功能21。适当的麻醉深度, 顺利和温和插管的动物气管插管, 和股动脉插管的血压监测没有出血是重要的, 在开始体外循环。如果体外循环前血流动力学不稳定, 成功率将显著降低。在体外循环期间, 必须通过调节储层容积来维持体外循环的流速。在体外循环术后60分钟, 由于炎症过程的血流动力学不稳定, 需要仔细添加体积 (1-3 毫升生理盐水)。然而, 过量胶体量的增加会导致贫血, 从而导致不稳定的病情。体外循环后, 体外循环回路中的血液应在30分钟内轻轻地返回老鼠. 体外循环后不稳定意味着有潜在的发病率, 如四肢或肠道缺血。
适当的麻醉深度是降低死亡率和发病率的一个提示。吸入麻醉剂为麻醉的深度和持续时间提供足够的控制。当我们对气管插管时, 老鼠必须在深度麻醉下才能失去反射。呼吸应该是有节奏的, 但不能被逮捕。呼吸骤停表明, 极深的麻醉、低血压和/或即将死亡, 导致全身炎症。反之, 麻醉不足会给动物带来更多的压力。在体外循环期间, 动物的身体运动是至关重要的减少, 因为可能意外清除套管。在手术过程中需要通过监测心率、动脉压力和胸壁运动来控制异氟醚蒸发器。在体外循环开始时, 异氟醚的血液浓度可能会因引物容积而减少。额外腹腔戊巴比妥可能允许稳定的麻醉深度。
大鼠术后感染会引起生理参数的大量变化22。长期生存模式要求严格维护的无菌条件在过程中。一个独立的房间和一个干净的工作区域的无菌程序是可取的。手术现场和器械应在使用前用70% 酒精或季铵盐消毒。任何可能影响污染的材料, 如套管、溶液和缝合材料, 都必须处于无菌包装中。在体外循环期间, 用湿润的小纱布覆盖手术场可防止这些部位受到潜在的污染。在关闭伤口前, 清除伤口的血液, 用无菌水冲洗, 以避免伤口感染。在我们的经验, 手术后没有伤口感染发生24小时。
这种体外循环模型的局限性在于不能完全模仿临床情况。首先, 这种心脏跳动模型表现出与心脏外科心脏手术不同的脉动流。其次, 该模型可进行无开胸手术, 不能充分再现中位开胸术的临床体外循环, 导致手术过程中大出血。
总之, 这种恢复体外循环模型允许研究早期和长期多器官损害与体外循环有关。该模型的主要优点包括通过输血、vasopressors 或强心剂剂减少可能的偏差。该模型适合于探讨预防体外循环多脏器损伤的治疗策略。

在 1953年, 约翰·小长臂猿成功地使用了 CPB1进行了第一次心脏手术, 随后它成为心血管外科的基本形态。虽然这些技术和设备已大为完善, 但与体外循环有关的多脏器并发症仍会损害心血管手术的结果, 并可能影响术后发病率和死亡率2。与体外循环有关的器官损害是由免疫系统活化和促炎细胞因子的合成、赞美活化和氧自由基的产生而引起的2。然而, 它的病理生理学尚未完全阐明。
动物模型综述的临床应用, 使对体外循环期间和之后的病理生理过程的澄清;这可以促进临床前研究, 以制定战略, 以避免这些并发症。自从鲍佩维奇et al。第一次报告了鼠体外循环模型在 1967年3, 大鼠体外循环模型已经完善, 并已逐渐取代大型动物模型, 由于更大的成本效益, 方便的实验过程, 和大量的测试方法在遗传和蛋白质水平。此外, 近亲繁殖的大鼠可以遗传相同, 减少可能的生物偏见。
Fabre et al。首先建立了恢复模型, 允许对 CPB 的长期多器官后遗症进行研究4。这种简单的生存模式的优点是灵活性 (体外循环流量和持续时间), 稳定的生命条件, 和重现性的系统性炎症。大鼠体外循环模型已成为研究预防体外循环5中多脏器损伤的治疗策略的关键, 最近开发了各种模拟体外循环临床情况的模型。De 兰格et al。建立了心脏骤停模型, 可用于描述与心肌损伤有关的酶、遗传和组织学反应7。彼得斯et al。采用小型化体外循环模型进行心肌梗死和控制再灌注, 通过局灶性缺血再灌注损伤分析心脏功能障碍8。Jungwirth et al。首先建立了深低温循环骤停 (DHCA) 模型, 它可以阐明 DHCA 的全球缺血再灌注损伤, 支持潜在神经保护策略6。研究使用 DHCA 调查体温过低, 再灌注和/或溶血触发信号事件的影响9。深低温可能会影响各种酶和通路的活化和失活, 而机制仍然未知的10。另一方面, 心脏骤停模型或心脏缺血模型必须用于研究缺血再灌注心脏损伤。这些不同的大鼠体外循环模型, 高度重述人类体外循环可能揭示与体外循环有关的病理过程, 并有助于缓解与体外循环相关的并发症。
该协议说明了一个简单的体外循环模型, 没有输血或强心剂剂的大鼠。该模型可用于研究体外循环的长期多脏器后遗症。

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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rodent Ventilator 7025</td>
			<td style="width:140px;">Ugo Basile</td>
			<td align="right" style="width:145px;">7025</td>
			<td style="width:167px;">Ventilator</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">OxiQuant B</td>
			<td style="width:140px;">ENVITEC</td>
			<td style="width:145px;">46-00-0023</td>
			<td>Oxygen Sensor</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">CMA 450 Temperature Controller</td>
			<td style="width:140px;">CMA</td>
			<td align="right" style="width:145px;">8003759</td>
			<td>Temperature Controller</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CMA 450 Heating Pad</td>
			<td style="width:140px;">CMA</td>
			<td align="right" style="width:145px;">8003763</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CMA 450 Rectal Probe</td>
			<td style="width:140px;">CMA</td>
			<td align="right" style="width:145px;">8003761</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">DIN(8) to Disposable BP Transducer</td>
			<td style="width:140px;">ADInstruments</td>
			<td style="width:145px;">MLAC06</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Disposable BP Transducer</td>
			<td style="width:140px;">ADInstruments</td>
			<td style="width:145px;">MLT0670</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">IX-214 Data Recorder</td>
			<td style="width:140px;">iWorx Systems</td>
			<td style="width:145px;">IWX-214</td>
			<td>amplifier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LabScribe software</td>
			<td style="width:140px;">iWorx Systems</td>
			<td style="width:145px;"></td>
			<td>software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Roller pump</td>
			<td style="width:140px;">Furue Science</td>
			<td style="width:145px;">Model RP-VT</td>
			<td>pump</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Happy Cath</td>
			<td style="width:140px;">Medikit</td>
			<td style="width:145px;">EB 19G 4HCLs PP</td>
			<td>17-gauge multiorifice angiocatheter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SURFLO ETFE I.V. Catheter</td>
			<td style="width:140px;">Terumo</td>
			<td style="width:145px;">SR-OX2419CA</td>
			<td>24-gauge angiocatheter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Oxygenator</td>
			<td style="width:140px;">Mera</td>
			<td style="width:145px;">HPO-002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CPB circuit</td>
			<td style="width:140px;">Mera</td>
			<td style="width:145px;">custom-made</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hespander fluid solution</td>
			<td style="width:140px;">Fresenius Kabi</td>
			<td style="width:145px;">3319547A4035</td>
			<td>Hydroxyethyl starch</td>
		</tr>
	</tbody>
</table>

a recovery cardiopulmonary bypass model without transfusion or inotropic agents in rats

体外循环 (CPB) 在心血管手术中是必不可少的。尽管体外循环技术和设备的戏剧性改良, 多脏器并发症与长期体外循环仍然影响心血管手术的结果, 并可能恶化的术后发病率和死亡率。动物模型综述的临床应用可以澄清在体外循环期间发生的病理生理过程, 并促进临床前期研究, 以制定预防这些并发症的战略。大鼠体外循环模型是有利的, 因为他们更大的成本效益, 方便的实验过程, 丰富的测试方法在基因或蛋白质水平, 和遗传一致性。它们可用于调查免疫系统激活和促炎性细胞因子的合成, 赞美活化和氧自由基的产生。大鼠模型已被提炼, 并逐渐取代了大型动物模型。在这里, 我们描述一个简单的体外循环模型, 没有输血和/或强心剂剂的老鼠。这种恢复模式允许研究体外循环的长期多器官后遗症。

图 1显示整个 CPB 电路。该模型中的生理变量显示在图 2中, 包括直肠温度、平均动脉血压和心率。图 3显示了体外循环期间的动脉血气分析, 包括动脉氧的分压, 动脉二氧化碳的分压, 红细胞积血, 碱过量, 血清钾的表达, 氢的电位。在整个手术过程中, 心率和平均动脉压力是稳定的。由于吸血量引起的血液稀释, 在体外循环开始时直肠温度和红细胞压积减少。在体外循环期间, 由于膜氧合, 动脉氧的分压明显增高。有代表性的苏木精和伊红染色的肺部图像显示在图 4中, 其中包括 CPB 组的图像 (图 4B, D) 和假操作组 (图 4A, C) 和炮2的比率/不负担装卸2 在 CPB 后与基线比较(图 4 E).对于间质水肿、炎症细胞浸润和出血, 体外循环组有显著性差异。图 5显示了体外循环后肿瘤坏死因子α (TNF α)、interleukin-6 (IL-6) 和高机动性组框 1 (HMGB1) 水平的血清浓度。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56986/56986fig1.jpg"> 图 1: 体外循环电路.整个 CPB 电路包括一个储层 (8 毫升), 一个膜肺与3.3 毫升的启动量, 和一个滚筒泵。所有部件通过聚氯乙烯管连接, 包括静脉引流线 (外径), 3.3 毫米; 内径 (ID), 2 毫米; 约1.4 毫升), 动脉线 (OD, 2 毫米;ID, 1.2 毫米;约1.2 毫升) 和一管附加泵辊 (OD, 6.6 毫米;ID, 4.5 毫米;约2.8 毫升)。总吸水量为11毫升, 包括储层约3毫升。<a href="//cloudflare.jove.com/files/ftp_upload/56986/56986fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56986/56986fig2.jpg"> 图 2: 生理变量.体外循环期间和术后的生理变量, 包括 (A) 直肠温度, (B) 平均动脉血压, (C) 心率 (n = 6)。指出了点和误差条的平均值和标准差。体外循环, 心肺旁路;前, 体外循环前;体外循环后的体外循环 x、x 分钟;后 x, x 分钟后, 体外循环结束。<a href="//cloudflare.jove.com/files/ftp_upload/56986/56986fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56986/56986fig3.jpg"> 图 3: 动脉血气分析.体外循环期间及术后动脉血气分析。(A) 动脉氧的分压, (B) 动脉二氧化碳的部分压力 (C) 红细胞压积, (D) 基数过剩, (E) 血清钾的表达, (F) 氢的电位 (n = 6)。指出了点和误差条的平均值和标准差。体外循环, 心肺旁路;前, 体外循环前;体外循环后的体外循环 x、x 分钟;后 x, x 分钟后, 体外循环结束。<a href="//cloudflare.jove.com/files/ftp_upload/56986/56986fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56986/56986fig4.jpg"> 图 4: 肺部组织学分析具有代表性的苏木精和伊红染色图像的假操作组 (A, C)和心肺旁路执行组 (B, D)。体外循环后4小时。呼吸窘迫的严重性与水肿和出血的病理表现程度相关。刻度条;50µm (A, B), 1 毫米 (C, D)。(E) 与假组和对照组 (每组中的 n = 6) 相比, CPB 后的2/不负担装卸2 的比率。指出了点和误差条的平均值和标准差。*p < 0.01 与假组。体外循环, 心肺旁路;炮2, 动脉氧分压;不负担装卸2, 激发氧的分数;后 x, x 分钟后, 体外循环结束。<a href="//cloudflare.jove.com/files/ftp_upload/56986/56986fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56986/56986fig5.jpg"> 图 5: 评估血清炎症反应.血清浓度 (A) 肿瘤坏死因子α (TNF α), (B) interleukin-6 (IL-6), 和 (C) 高机动性组框 1 (HMGB1) 水平后体外循环。n = 5 为每组: 假组和体外循环组, 大鼠进行体外循环手术。指出了点和误差条的平均值和标准差。*p < 0.05, †p < 0.001 与假组。体外循环, 心肺旁路;后 x, x 分钟后, 体外循环结束。<a href="//cloudflare.jove.com/files/ftp_upload/56986/56986fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about A Recovery Cardiopulmonary Bypass Model Without Transfusion or Inotropic Agents in Rats at JoVE.com

A Recovery Cardiopulmonary Bypass Model Without Transfusion or Inotropic Agents in Rats

无输血或强心剂剂的大鼠心肺旁路移植模型

在这里, 我们提出一个协议来描述一个简单的恢复心肺旁路模型没有输血或强心剂剂的大鼠。该模型可用于体外循环术后长期多脏器后遗症的研究。

Cardiopulmonary bypass (CPB) is indispensable in cardiovascular surgery. Despite the dramatic refinement of CPB technique and devices, multi-organ ...

a-recovery-cardiopulmonary-bypass-model-without-transfusion-or

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

7.8K 次观看.  Kyoto University. 在这里, 我们提出一个协议来描述一个简单的恢复心肺旁路模型没有输血或强心剂剂的大鼠。该模型可用于体外循环术后长期多脏器后遗症的研究。

视频: A Recovery Cardiopulmonary Bypass Model Without Transfusion or Inotropic Agents in Rats

Orthotopic Hind-Limb Transplantation in Rats

Composite tissue allotransplantation (CTA) now represents a valid therapeutic option after the loss of a hand, forearm or digits and has become a novel therapeutic entity in reconstructive surgery. However, long term high-dose multi-drug immunosuppressive therapy is required to ensure graft survival, bearing the risk of serious side effects which halters broader application. Further progression in this field may depend on better understanding of basic immunology and ischemia reperfusion injury in composite tissue grafts.
To date, orthotopic hind limb transplantation in rats has been the preferred rodent model for reconstructive transplantation (RT), however, it is an extremely demanding procedure that requires extraordinary microsurgical skills for reattachment of vasculature, bones, muscles and nerves.
We have introduced the vascular cuff anastomosis technique to this model, providing a rapid and reliable approach to rat hind limb transplantation. This technique simplifies and shortens the surgical procedure and enables surgeons with basic microsurgical experience to successfully perform the operation with high survival and low complication rates. The technique seems to be well suited for immunological as well as ischemia reperfusion injury (IRI) studies.

Here we describe the orthotopic rat hind-limb transplantation procedure, which seems to be the gold standard in vivo model for composite tissue allotransplantation research.

原位大鼠后肢移植

Composite tissue allotransplantation (CTA) now represents a valid therapeutic option after the loss of a hand, forearm or digits and has become a novel ...

科研

免疫与感染

Diagnosis of Ecto- and Endoparasites in Laboratory Rats and Mice

Internal and external parasites remain a significant concern in laboratory rodent facilities, and many research facilities harbor some parasitized animals. Before embarking on an examination of animals for parasites, two things should be considered. One: what use will be made of the information collected, and two: which test is the most appropriate. Knowing that animals are parasitized may be something that the facility accepts, but there is often a need to treat animals and then to determine the efficacy of treatment. Parasites may be detected in animals through various techniques, including samples taken from live or euthanized animals. Historically, the tests with the greatest diagnostic sensitivity required euthanasia of the animal, although PCR has allowed high-sensitivity testing for several types of parasite. This article demonstrates procedures for the detection of endo- and ectoparasites in mice and rats. The same procedures are applicable to other rodents, although the species of parasites found will differ.

This article describes various procedures for screening rats and mice to detect endo- or ectoparasitism. Several diagnostic assays will be demonstrated, both those suitable for use on live animals and those used after euthanasia of the animal. Photographs to aid in identification of rat and mouse parasites will be included.

Ecto和实验室大鼠和小鼠体内寄生虫的诊断

Internal and external parasites remain a significant concern in laboratory rodent facilities, and many research facilities harbor some parasitized ...

Reverse Genetics Mediated Recovery of Infectious Murine Norovirus

Human noroviruses are responsible for most cases of human gastroenteritis (GE) worldwide and are recurrent problem in environments where close person-to-person contact cannot be avoided 1, 2. During the last few years an increase in the incidence of outbreaks in hospitals has been reported, causing significant disruptions to their operational capacity as well as large economic losses. The identification of new antiviral approaches has been limited due to the inability of human noroviruses to complete a productive infection in cell culture 3. The recent isolation of a murine norovirus (MNV), closely related to human norovirus 4 but which can be propagated in cells 5 has opened new avenues for the investigation of these pathogens 6, 7. 
MNV replication results in the synthesis of new positive sense genomic and subgenomic RNA molecules, the latter of which corresponds to the last third of the viral genome (Figure 1). MNV contains four different open reading frames (ORFs), of which ORF1 occupies most of the genome and encodes seven non-structural proteins (NS1-7) released from a polyprotein precursor. ORF2 and ORF3 are contained within the subgenomic RNA region and encode the capsid proteins (VP1 and VP2, respectively) (Figure 1). Recently, we have identified that additional ORF4 overlapping ORF2 but in a different reading frame is functional and encodes for a mitochondrial localised virulence factor (VF1) 8.
Replication for positive sense RNA viruses, including noroviruses, takes place in the cytoplasm resulting in the synthesis of new uncapped RNA genomes. To promote viral translation, viruses exploit different strategies aimed at recruiting the cellular protein synthesis machinery 9-11. Interestingly, norovirus translation is driven by the multifunctional viral protein-primer VPg covalently linked to the 5' end of both genomic and subgenomic RNAs 12-14. This sophisticated mechanism of translation is likely to be a major factor in the limited efficiency of viral recovery by conventional reverse genetics approaches. 
Here we report two different strategies based on the generation of murine norovirus-1 (referred to as MNV herewith) transcripts capped at the 5' end. One of the methods involves both in vitro synthesis and capping of viral RNA, whereas the second approach entails the transcription of MNV cDNA in cells expressing T7 RNA polymerase. The availability of these reverse genetics systems for the study of MNV and a small animal model has provided an unprecedented ability to dissect the role of viral sequences in replication and pathogenesis 15-17.

Noroviruses are a major cause of gastroenteritis yet molecular techniques for their characterisation are still relatively new. Here we report two different reverse genetics approaches for the efficient recovery of murine norovirus (MNV), the only member of this genus which can be propagated in cell culture.

反向遗传学感染小鼠诺沃克病毒介导的恢复

Human noroviruses are responsible for most cases of human gastroenteritis (GE) worldwide and are recurrent problem in environments where close ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

中性粒细胞分离和分析，以确定他们的淋巴瘤细胞的敏感性对治疗药物的作用

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or A. baumannii

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, preferably in models which mimic the intended clinical indication. A method for inducing robust lung infections in immunocompetent rats and mice is described which allows for the assessment of treatments in a model of serious pneumonia caused by S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae or A. baumannii. Animals are anesthetized, and an agar-based inoculum is deposited deep into the lung via nonsurgical intratracheal intubation. The resulting infection is consistent, reproducible, and stable for at least 48 h and up to 96 h for most isolates. Studies with marketed antibacterials have demonstrated good correlation between in vivo efficacy and in vitro susceptibility, and concordance between pharmacokinetic/pharmacodynamic targets determined in this model and clinically accepted targets has been observed. Although there is an initial time investment when learning the technique, it can be performed quickly and efficiently once proficiency is achieved. Benefits of the model include elimination of the neutropenic requirement, increased robustness and reproducibility, ability to study more pathogens and isolates, improved flexibility in study design and establishment of a challenging infection in an immunocompetent host.

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or  A. baumannii

A method for establishing lung infections in immunocompetent rodents is described. With proficiency, this method can be performed quickly and easily to induce stable infection with many isolates of S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae and A. baumannii.

在免疫鼠害的鲁棒性肺炎模型评价抗菌功效反对<i&gt;秒。肺炎</i&gt;<i&gt;小时。流感</i&gt;<i&gt; K。肺炎</i&gt;<em&gt;点。铜绿假单胞菌</em&gt;或<em&gt;一个。鲍曼不动杆菌</em

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, ...

Optimized Interferon-gamma ELISpot Assay to Measure T Cell Responses in the Guinea Pig Model after Vaccination

The guinea pig has played a pivotal role as a relevant small animal model in the development of vaccines for infectious diseases such as tuberculosis, influenza, diphtheria, and viral hemorrhagic fevers. We have demonstrated that plasmid-DNA (pDNA) vaccine delivery into the skin elicits robust humoral responses in the guinea pig. However, the use of this animal to model immune responses was somewhat limited in the past due to the lack of available reagents and protocols to study T cell responses. T cells play a pivotal role in both immunoprophylactic and immunotherapeutic mechanisms. Understanding T cell responses is crucial for the development of infectious disease and oncology vaccines and accommodating delivery devices. Here we describe an interferon-gamma (IFN-&#947;) enzyme-linked immunospot (ELISpot) assay for guinea pig peripheral blood mononuclear cells (PBMCs). The assay enables researchers to characterize vaccine-specific T-cell responses in this important rodent model. The ability to assay cells isolated from the peripheral blood provides the opportunity to track immunogenicity in individual animals.

The development of the interferon-gamma ELISpot assay for guinea pig PBMCs allows the characterization of T-cell responses in this highly relevant model for studying infectious diseases. We have applied the assay to measure T cell responses associated with intradermal delivery of DNA vaccines.

优化干扰素伽隆弹性体检测在几内亚猪模型接种疫苗后 t 细胞反应

The guinea pig has played a pivotal role as a relevant small animal model in the development of vaccines for infectious diseases such as tuberculosis, ...

A Precise Pathogen Delivery and Recovery System for Murine Models of Secondary Bacterial Pneumonia

Secondary bacterial pneumonias following influenza infections consistently rank within the top ten leading causes of death in the United States. To date, murine models of co-infection have been the primary tool developed to explore the pathologies of both the primary and secondary infections. Despite the prevalence of this model, considerable discrepancies regarding instillation procedures, dose volumes, and efficacies are prevalent among studies. Furthermore, these efforts have been largely incomplete in addressing how the pathogen may be directly influencing disease progression post-infection. Herein we provide a precise method of pathogen delivery, recovery, and analysis to be used in murine models of secondary bacterial pneumonia. We demonstrate that intratracheal instillation enables an efficient and accurate delivery of controlled volumes directly and evenly into the lower respiratory tract. Lungs can be excised to recover and quantify the pathogen burden. Following excision of the infected lungs, we describe a method to extract high quality pathogen RNA for subsequent transcriptional analysis. This procedure benefits from being a non-surgical method of delivery without the use of specialized laboratory equipment and provides a reproducible strategy to investigate pathogen contributions to secondary bacterial pneumonia.

Here, we present methods to improve secondary bacterial pneumonia studies by providing a non-invasive route of instillation into the lower respiratory tract followed by pathogen recovery and transcript analysis. These procedures are reproducible and can be performed without specialized equipment such as cannulas, guide wires, or fiber optic cables.

二次细菌性肺炎的鼠种模型的精确病原体输送与恢复系统

Secondary bacterial pneumonias following influenza infections consistently rank within the top ten leading causes of death in the United States. To date, ...

Single-cell Screening Method for the Selection and Recovery of Antibodies with Desired Specificities from Enriched Human Memory B Cell Populations

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational methods, such as next generation sequencing (NGS), yield enormous sets of data on the antibody repertoire at the sequence level, functional data is required to identify which sequences are relevant for a particular antigen or set of antigens. Here, we describe a method to identify and recover individual antigen-specific antibodies from peripheral blood mononuclear cells (PBMCs) from a human blood donor. This method utilizes an initial enrichment of mature B cells and requires a combination of phenotypic cell markers and fluorescently-labeled protein to isolate IgG memory B cells via flow cytometry. The heavy and light chain variable regions are then cloned and re-screened. Although limited to the memory B cell compartment, this method takes advantage of flow cytometry to interrogate millions of B cells and returns paired heavy and light chain sequences from a single cell in a format ready for expression and confirmation of specificity. Antibodies recovered with this method can be considered for therapeutic potential, but can also link specificity and function with bioinformatic approaches to assess the B cell repertoire within individuals.

The BSelex method to identify and recover individual antigen-specific antibodies from human peripheral blood mononuclear cells combines flow cytometry with single cell PCR and cloning.

从富足人类记忆B细胞群中选择和恢复具有所需特异性的抗体的单细胞筛选方法

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational ...

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are cleared from the human lung alveoli by being phagocytosed by innate monocytes and/or neutrophils. This protocol offers a fast and reliable measurement of phagocytosis by flow cytometry using fluorescein isothiocyanate (FITC)-labeled conidia for co-incubation with human leukocytes and subsequent counterstaining with an anti-FITC antibody to allow discrimination of internalized and cell-adherent conidia. Major advantages of this protocol are its rapidness, the possibility to combine the assay with cytometric analysis of other cell markers of interest, the simultaneous analysis of monocytes and neutrophils from a single sample and its applicability to other cell wall-bearing fungi or bacteria. Determination of percentages of phagocytosing leukocytes provides a means to microbiologists for evaluating virulence of a pathogen or for comparing pathogen wildtypes and mutants as well as to immunologists for investigating human leukocyte capabilities to combat pathogens.

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

This protocol provides a fast and reliable method to quantitatively measure phagocytosis of Aspergillus fumigatus conidia by human primary phagocytes using flow cytometry and to discriminate phagocytosis of conidia from mere adhesion to leukocytes.

利用流细胞测定人类白细胞测定阿斯珀吉鲁斯紫锥体烟曲的噬菌体

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are ...

Visualization and Quantification of High-Dimensional Cytometry Data using Cytofast and the Upstream Clustering Methods FlowSOM and Cytosplore

The complexity of data generated by mass cytometry has necessitated new tools to rapidly visualize analytic outcomes. Clustering methods like Cytosplore or FlowSOM are used for the visualization and identification of cell clusters. For downstream analysis, a newly developed R package, Cytofast, can generate a rapid visualization of results from clustering methods. Cytofast takes into account the phenotypic characterization of cell clusters, calculates the cell cluster abundance, then quantitatively compares groups. This protocol explains the applications of Cytofast to the use of mass cytometry data based on modulation of the immune system in the tumor microenvironment (i.e., the natural killer [NK] cell response) upon tumor challenge followed by immunotherapy (PD-L1 blockade). Demonstration of the usefulness of Cytofast with FlowSOM and Cytosplore is shown. Cytofast rapidly generates visual representations of group-related immune cell clusters and correlations with immune system composition. Differences are observed in the clustering analysis, but separation between groups are visible with both clustering methods. Cytofast visually shows the patterns induced by PD-L1 treatment that include a higher abundance of activated NK cell subsets, expressing a higher intensity of activation markers (i.e., CD54 or CD11c).

Cytofast is a visualization tool used to analyze output from clustering. Cytofast can be used to compare two clustering methods: FlowSOM and Cytosplore. Cytofast can rapidly generate a quantitative and qualitative overview of mass cytometry data and highlight the main differences between different clustering algorithms.

使用Cytofast和上游聚类方法流和细胞溅的高维细胞测量数据的可视化和量化

The complexity of data generated by mass cytometry has necessitated new tools to rapidly visualize analytic outcomes. Clustering methods like Cytosplore ...