Name: real-time assessment of spinal cord microperfusion in a porcine model of ischemia/reperfusion
Uploaded: 2020-12-10T14:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Real-Time Assessment of Spinal Cord Microperfusion in a Porcine Model of Ischemia/Reperfusion at JoVE.com

作者要感谢汉诺威医学院动物研究所的Lena Brix，V.M.D，以及德国汉堡 - 埃彭多夫大学医学中心动物护理研究设施Jutta Dammann女士提供术前和围手术期动物护理以及他们在动物处理方面的技术援助。作者还要感谢卢森堡基希贝格医院血管外科的Daniel Manzoni博士的技术援助。

该研究已获得汉堡市动物护理和使用政府委员会的批准（参考编号:60/17）。根据"实验动物的护理和使用指南"（NIH出版物第86-23号，2011年修订）以及FELASA建议和实验根据ARRIVE指南24，25进行护理。这项研究是一项急性试验，所有动物在方案结束时都接受了安乐死。
注:该研究是在六头体重约40公斤的三个月大的雄猪和雌猪（德国地方猪）中进行的。在实验前至少7天将动物带到动物护理设施，并根据动物福利建议进行饲养。随意为动物提供食物和水，并由负责的兽医定期评估其健康状况。在实验前保持了12小时的空腹时间。动物的整个实验过程和处理由负责的兽医监督。
1. 麻醉诱导及维持麻醉
<ol><li>对于麻醉诱导和麻醉维持，对动物进行预用，并使用肌内注射进行深度镇静，然后静脉注射，如有必要，进行气管插管。此后，通过使用挥发性麻醉剂与连续阿片类药物应用的组合来诱导和维持麻醉，并辅以额外的阿片类药物推注。</li>
	<li>肌内注射氯胺酮20mg·kg-1、氮杂松4mg·kg-1和咪达唑仑0.1mg·kg-1，用于术前和镇静。</li>
	<li>将静脉导管置于耳静脉中，确保正确固定，并通过快速施用10 mL盐水来评估功能。</li>
	<li>将动物置于加热毯上的仰卧位，以防止热量损失。</li>
	<li>通过心电图（ECG）和脉搏血氧饱和度法建立基本监测，以监测动物的心肺状态，并将其连接到基本监测硬件。</li>
	<li>通过猪形面罩给予15 L·min-1 氧气进行预氧。</li>
	<li>必要时静脉注射0.1mg·kg-1% 丙泊酚，并进行气管插管。</li>
	<li>通过潮气末二氧化碳图和听诊确保正确放置，给予0.1mg•kg-1的泛库溴铵，并确保气管插管的正确固定。</li>
	<li>使用麻醉机使用10 mL·kg-1体重-1 的潮气量，10cmH2O的呼气末正压和0.3的吸入氧气（FiO2）的分数建立体积控制通气。调整呼吸机频率，以保持呼气末二氧化碳张力 （etCO2）为 35-45 mmHg。</li>
	<li>引入胃管，进行胃液抽吸，正确固定管子，然后将其连接到收集袋。小心地关闭动物的眼睛，以防止麻醉期间眼睛干燥。</li>
	<li>通过连续输注芬太尼（10μg·kg-1·h -1）和七氟醚（3.0%过期浓度，由蒸汽输送）来维持麻醉。通过仔细观察生命体征和通气参数以及在整个治疗过程中没有任何运动来确保足够的麻醉水平，特别注意手术刺激的阶段。如果有任何疼痛或痛苦的迹象，给予额外推注剂量的芬太尼（50μg）。 注意:确保在整个过程中有在动物麻醉方面有经验的研究人员在场，并使用经验丰富的兽医的监督来确保适当的麻醉。</li>
	<li>给予基线输注速率为10 mL·kg-1·h-1平衡晶体，以补偿麻醉，手术准备和执行实验方案期间的液体损失。使用液体加热器防止热量损失。</li>
	<li>用肥皂水轻轻清洁猪皮。使用含有聚维酮碘的皮肤消毒液来减少皮肤污染。使用无菌手套进行手术准备。施用300mg克林霉素作为抗菌预防，6小时后重复剂量。</li>
</ol>2. 探头放置
<ol><li>将动物放在正确的横向位置，并弯曲动物的背部以扩大椎骨之间的空间。</li>
	<li>手术暴露椎旁区域以准备棘突和椎弓（图1A）。</li>
	<li>将血管14 G外周静脉导管正中正中置于胸椎（Th）13/14或腰椎（L）1/2水平的两个椎弓之间（图1B）。</li>
	<li>取出针头，将激光/多普勒针探头插入静脉导管（图1C），并通过连接到指定的硬件和软件来测试信号质量。确保有稳定的信号，具有中度脉动。</li>
	<li>小心地用缝合线固定探头（图1D），并使用衬垫防止探头脱臼或扭结。</li>
	<li>对于经皮放置脑脊液引流以测量和控制脑脊压，确定L 4/5或L 5/6的水平，用引入针刺穿皮肤和皮下间隙，并取出嵌体针。</li>
	<li>将充满盐水的注射器放在针头上，并在充满液体的注射器上以恒定的压力小心地引入针头。</li>
	<li>一旦感觉到阻力丧失是硬膜外位置的证据，请重新引入嵌体针，并进一步引入2-3mm的针头以刺穿硬脑膜并取出嵌体针头。</li>
	<li>通过快速滴落清澈的液体来验证鞘内位置。引入最深 20 cm 的引流口，安装鲁尔锁适配器，并通过小心抽吸液体来验证位置。</li>
	<li>用缝合线小心地固定引流，并将其连接到脑脊液引流系统。</li>
	<li>将头骨暴露在左耳后面，并使用6毫米的钻孔附件小心地对皮肤进行钻孔钻孔。</li>
	<li>将第二个激光多普勒探针直接引入大脑。小心地用缝合线固定探头，并通过连接到指定的硬件和软件来测试信号质量。同样，确保有一个稳定的信号，具有中度的脉动性。</li>
	<li>断开所有探头，小心地将动物置于仰卧位置，确保探头位置不受影响。确保至少有4-5名研究人员执行此操作。</li>
	<li>重新连接探头，然后重新检查信号质量。</li>
	<li>将激光多普勒硬件的输出通道连接到放大器和同步采集硬件和软件，以额外记录激光/多普勒通量与大血流动力学信号同时。</li>
	<li>通过 2 点校准按单位校准通量 （PU）。<ol>
<li>按 Enter 打开菜单，然后选择 模拟输出设置。
</li>
		<li>使用显示的转换因子（5.0 V = 1000 PU）通过2点校准校准助焊剂，以便与同步采集软件一起使用。</li>
		<li>选择 返回 以返回上一个菜单，然后选择 测量 以继续测量。</li>
		<li>打开同步采集软件。从"设置"菜单中选择"所有输入"零。将所有输入与所使用的设备和探头连接。</li>
		<li>通过单击通量通道的下拉菜单对助焊剂执行2点校准。选择2 点校准。将单位转换设置为开，然后选择BPU作为单位。对于点 1，将0 V设置为0 BPU。对于第 2 点，将5.0 V设置为1000 BPU。选择"所有数据"和"新数据"的设置单位。按"确定"关闭菜单。</li>
	</ol></li>
	<li>开始连续脑脊液引流，目标压力为 10 mmHg，引流量为 20 mL·h-1。</li>
</ol>3. 导管放置
<ol><li>暴露两股动脉。</li>
	<li>固定右股动脉的远端部分，使用血管环暂时阻塞动脉近端腔，使用Potts'剪刀对血管进行2mm切割，并引入导丝。</li>
	<li>进一步引入导丝，确保无电阻插入，避免导线任何扭结;将导管引入导线。</li>
	<li>用缝合线固定导管。</li>
	<li>通过抽吸动脉血确保正确的位置，在正确连接到血压和经心肺监测硬件和软件后，通过血气分析和动脉信号测量进行验证。</li>
	<li>在左股动脉上放置一个5mm的流量探头，并通过连接到流量计来测试信号质量。</li>
	<li>用缝合线缝合两个腹股沟。</li>
	<li>暴露右侧颈动脉以及右侧颈内静脉，以放置 8 个 Fr. 引入鞘。</li>
	<li>对于导管放置，请按照3.2-3.4中描述的相同方式进行。</li>
	<li>将颈动脉导流鞘的侧腔连接到用于动脉压测量的基本压力监测和肺热稀释硬件。</li>
	<li>将压尖导管引入升主动脉，并通过连接到放大器和同步采集硬件和软件来验证位置。</li>
	<li>将 Swan-Ganz 肺动脉导管通过肺动脉中的静脉鞘放置，方法是在 20 cm 深度用空气充气球囊并轻轻插入，直到血流动力学曲线中出现楔形压力。放气球囊，将导管向后拉 2 cm。确保满足肺动脉压的信号质量。将热敏电阻连接到基本压力监测和肺热稀释硬件。</li>
	<li>使用超声引导将 12 Fr. 5-腔中心静脉导管经皮放置给药和右颈外静脉中心静脉压测量。使用 6 步法进行超声放置38
</li>
	<li>将导管的远端腔连接到血压和经心肺监测硬件和软件。将所有药物和输液切换到中心静脉导管。使用不同的腔体进行镇痛药、液体和儿茶酚胺，并在容量加载步骤中留出大腔用于胶体给药。</li>
</ol>4. 手术准备
<ol><li>进行小型剖腹手术，调动膀胱，插入导尿管进行尿液引流，用生理盐水给球囊充气，并用袋缝合线固定导管。</li>
	<li>将导管连接到尿液收集袋，显示以 mL 为单位的尿量。</li>
	<li>将FiO2 增加到1.0，静脉内重新给予0.1mg·kg-1 苞库溴铵。</li>
	<li>通过使用电烙术准备到胸骨进行正中胸骨切开术。轻轻地从周围组织中解剖胸骨。进行胸骨后压迫以防止受伤。</li>
	<li>停止通气，用摆动的锯子将骨头分开。继续通风并将 FiO2 降至 0.3。使用电烙术减少出血，并用骨蜡密封胸骨。</li>
	<li>小心地调动左肺的顶点，并分割横膈膜的左侧外侧部分，以方便手术暴露。</li>
	<li>通过轻轻地缩回左肺，将降主动脉近端暴露在乳糜泻干上，确保通气不受干扰，避免左肺外伤（图2A）并分割周围组织（图2B）。如果需要血流动力学稳定，施用7mL·kg-1 羟乙基淀粉胶体。</li>
	<li>在下主动脉周围放置一个支撑，以确保正确曝光（图2C）。</li>
	<li>在下降的胸主动脉周围安装一个流动探头（图2D）。通过连接到流量模块和同步采集硬件和软件，确保适当的信号质量。如果需要，使用接触凝胶来改善信号质量。</li>
	<li>在下降的主动脉周围安装一个血管环，远端到流动探头，以标记主动脉交叉夹紧的区域。</li>
</ol>5. 评估和数据采集
<ol><li>将所有导管和水平导管清零，使用放置在右侧心房水平的充满液体的管路。</li>
	<li>放置针头心电图电极，并将其连接到同步采集硬件和软件。</li>
	<li>经心肺热稀释以及主动脉血流和压力测量的评估以前已有描述34。</li>
	<li>对于使用肺动脉热稀释的心输出量测量，用10 mL冷盐水进行3次注射，并注意基本监测硬件显示的平均值。</li>
	<li>只需按" 开始"键即可启动激光多普勒软件，然后通过仔细地将步骤标记为 M0 到 M5为每个测量步骤设置标记。</li>
</ol>6. 实验方案
<ol><li>执行基线测量 （M0）。</li>
	<li>使用7mL·kg-1 羟乙基淀粉胶体的体积加载步骤进行血流动力学优化。使用加压输液在5分钟内执行每个体积加载步骤。完成每个体积加载步骤后，等待5分钟以平衡。开始容量负荷，直到心输出量增加<15%。</li>
	<li>完成血流动力学优化后重复测量（M1）。</li>
	<li>通过在标记区域放置主动脉钳，诱导缺血/再灌注共 48 分钟乳糜泻上主动脉交叉钳夹。</li>
	<li>在研究方案期间，以1-，2-，5-，10-和30分钟间隔的升序应用主动脉夹紧，以提高动物的存活率。</li>
	<li>每个间隔后，最多 5 分钟或股动脉正常化后继续主动脉交叉钳夹。</li>
	<li>对下腔静脉进行手动流入阻塞，以防止血压升高>平均动脉压100 mmHg。</li>
	<li>如果需要，在钳夹阶段推注去甲肾上腺素或肾上腺素，以防止平均动脉压降至40 mmHg以下。</li>
	<li>在再灌注（M2）之前的30分钟夹紧间隔结束时重复测量。</li>
	<li>逐渐打开夹子，确保血流动力学稳定。如果血压下降太快，请关闭夹子并允许稳定。</li>
	<li>给予7 mL·kg-1 羟乙基淀粉胶体以及额外的推注10-20μg去甲肾上腺素和/或肾上腺素以稳定。如果pH值降至7.1以下，则给予2 mL kg-1 的8.4%碳酸氢钠。确保适当调整呼吸频率，以确保正常碳酸血症。</li>
	<li>再灌注后1小时重复测量（M3）。</li>
	<li>重复血流动力学优化，如6.2下所述，并重复测量（M4）。</li>
	<li>在诱导缺血/再灌注（M5）后4.5小时进行最终测量。</li>
</ol>7. 安乐死
<ol><li>静脉注射40 mmol氯化钾用于安乐死，以诱发心室颤动和心搏停止。</li>
	<li>终止通气并取出所有导管。</li>
</ol>8. 器官摘取
<ol><li>将动物置于俯卧位置，并取出针头探头和引流。</li>
	<li>通过皮肤切口暴露脊柱，并使用手术刀和镊子去除肌肉组织。</li>
	<li>使用振荡锯将椎弓正中侧分开两侧，并通过小心地将棘突侧向松动其余连接来移除椎骨的背侧部分。</li>
	<li>用镊子小心地将脊髓从尾端抬到颅端，用手术刀切开脊神经，切除脊髓。</li>
	<li>将脊髓储存在4%福尔马林中，直到进一步用于组织病理学评估或微球定量。</li>
</ol>9. 统计分析
<ol><li>使用统计软件。</li>
	<li>如有必要，通过检查直方图和对数变换变量来确保正态分布。</li>
	<li>将因变量（脊髓通量、心输出量、心率、每搏量、收缩动脉压、平均动脉压、舒张动脉压、中心静脉压、全身血管阻力）以及根据需要用荧光微球评估的上脊髓和下部微灌注）应用于一般线性混合模型分析，使用常规 GENLINMIXED 进行具有恒等链接函数的连续数据。</li>
	<li>使用基线调整。</li>
	<li>为可变基线和测量点指定具有固定效应的模型。将测量点视为动物体内的重复测量值。</li>
	<li>报告每个参数的测量点的固定效应的 p 值。</li>
	<li>对于脊髓荧光微球分析，另外使用区域（下脊髓，上脊髓）作为区域和测量点之间的固定效应和相互作用，以评估区域和测量点之间的相互作用，并报告固定效应的p值以进行相互作用。</li>
	<li>计算测量点 M1-M5 处所有因变量的基线调整边际均值，置信区间 （CI） 为 95%，然后通过最小显著性差值检验进行成对比较。</li>
	<li>将变量表示为平均值（95% CI）。将动物重量表示为平均±标准差。</li>
	<li>显示未调整的 p 值。</li>
</ol>

Constantin J.C. Trepte因Maquet的演讲而获得荣誉奖。所有其他作者声明没有利益冲突。这项研究得到了欧洲麻醉学会2018年青年研究员启动资助的支持。

脊髓缺血诱发的SCI是主动脉修复的主要并发症，对患者的预后有巨大影响1，2，3，4，10，11，12。预防和治疗 SCI 的微循环靶向疗法是最有希望的。该协议为实时脊髓微循环评估提供了一种可重复的方法，并提供了评估缺血/再灌注条件下新型治疗方法对脊髓微循环的影响的能力。
在这个实验模型中有一些关键的方法学步骤。为了防止动物丢失，研究人员必须在麻醉技术（主动脉暴露期间脑脊液引流插入，超声血管通路和血流动力学治疗，主动脉交叉钳夹和再灌注）以及手术技术（胸骨切开术，血管暴露，下主动脉的手术暴露）方面经验丰富。插入脊髓针探头需要经验，深厚的解剖学知识和良好的技术技能。然而，根据我们的经验，学习曲线相当陡峭，大多数有经验的研究人员将在短时间内取得成功，尽管必须避免多次尝试以防止可能影响方法的脊髓损伤。
另一个关键步骤是从右侧位改为仰卧位，以防止脊髓针探针脱位或损伤。对于这种操作，建议4-5人，插入部位的适当填充至关重要，并且应谨慎行事，不要使探头脱臼。暴露降主动脉也需要一些关键步骤。必须调动左肺的顶点，以允许左肺轻轻地回缩以暴露手术区域。此外，应解剖隔膜的左外侧部分，以利于暴露。在主动脉准备期间，需要进行手术的研究人员与提供麻醉和血流动力学管理的研究人员之间的最佳沟通，以确保足够的心肺稳定性。在主动脉交叉钳夹期间，建议手动压迫下腔静脉以减少静脉回流。如果没有这种操作，可能会发生严重的后负荷增加，可能导致有害的心肌损伤39，40。
应谨慎地使用液体、血管加压药和正性肌力药物进行再灌注。在再灌注期间，会发生剧烈变化，可能导致严重的低血压，心律失常和循环衰竭41。然而，谨慎观察血流动力学行为，迅速启动干预措施，以及在这个关键阶段使用结构化和温和的表现可以防止动物的损失。此外，使用主动脉交叉钳夹的上升间隔，随后是时间段以改善再生，如方案中所使用的那样，诱导缺血预处理效应，增强再灌注期间的血流动力学稳定性42，43。
该模型除了提供大循环评估外，还提供了监测脊髓微循环的能力。由于在高危手术和危重患者中经常出现血流动力学连贯性的丧失，因此需要直接评估脊髓微循环13，30。舌下微循环通常用于替代感兴趣器官中的直接微循环评估44。然而，舌下微循环与重要器官之间的解离已被证明，强调了脊髓中直接微循环评估的价值，如实验模型45所示。最后，与荧光微球评估相比，该模型具有实时监测脊髓血流的优点，后者受到间歇使用和验尸分析的限制46。当查看缺血和再灌注诱导期间的示例记录时，可以最好地看到实时评估的影响，显示脊髓微灌注的快速变化。然而，应该考虑到激光多普勒探针插入脊髓可能导致脊髓的小而相当大的损伤。
由于脊髓的完整性可能会影响血流动力学参数，因此这可能是该方法的缺点。然而，使用激光多普勒技术来评估脊髓微灌注以前已经使用了47，48，49，50。此外，尽管我们没有观察到探针插入后的血流动力学变化，但我们不能排除该方法诱导的血流动力学效应。应该注意的是，使用微球注射也可能引起血流动力学改变，然而，这在大型动物中的重要性不大51。此外，探针插入可能会影响感觉或运动功能，因此，应谨慎使用感觉或运动诱发电位评估，并结合激光多普勒评估。
在这方面，微球注射技术可能是有利的。此外，这些技术不应用于慢性试验;然而，对于微球注射也是如此，微球注射仅限于急性试验，因为它们依赖于死后组织分析。大多数使用激光多普勒技术的研究都是在小动物身上进行的47，48，49，50在这里，我们描述了一种用于猪的技术，作为一种大型动物模型，可以促进转化为临床研究。旁正中体引入技术克服了猪体内大棘突的问题，这使得脊髓探针的正确放置变得复杂。此外，该技术的优点是不需要椎板切除术或去除硬脑膜组织，从而防止液体的持续损失。由于脑脊液压力对脊髓灌注32有巨大影响，该模型具有测量和优化脑脊液压力以及脊髓微灌注的优势，并将在今后的项目中解决脑脊液压力对脊髓微灌注的影响。
该协议有一些应该提到的限制。由于确切探针位置和较大脊髓血管的接近度的差异，动物之间脊髓通量的绝对值差异很大。因此，在比较值时应执行基线调整。然而，测量点之间的个体间差异是高度一致的，只要谨慎行事以避免在实验方案期间针头探头移动。此外，这项研究并不是作为激光多普勒和荧光微球方法之间的比较研究而设计的。考虑到动物的数量，我们没有对这两种方法进行相关性分析。
虽然这两种方法都表现出可比的行为，在缺血期间和再灌注后的恢复期间显着减少，但将来应使用适当设计的研究来解决这些方法的比较。然而，微球的使用还能够评估上脊髓微灌注的不同行为。此外，组织病理学分析显示，与其他脊髓缺血模型相比，只有中度脊髓坏死37。延长缺血的持续时间以及省略预处理措施可能会导致一些研究人员可能想要的更严重的变化。虽然我们仅评估了轻度组织病理学变化，但随着缺血持续时间的延长，这可能有所不同。在这方面，在方案终止之前的缺血/再灌注后较长时间也可能导致更严重的组织病理学变化。然而，该协议使再灌注后一小时的血流动力学稳定，而无需额外甚至连续的正性肌力或血管加压药应用。
对于不同血流动力学干预的评估，该模型提供了最佳条件。虽然我们使用液体优化作为血流动力学干预的一个例子，但可以使用该方法评估其他方法。虽然该协议在缺血/再灌注模型中提供微循环评估，但缺血的持续时间限制了再灌注前缺血期间治疗方法的评估。此外，在缺血期间，血流动力学变化发生变化（例如，高血压、低血压、心动过速、心动过缓以及心律失常）。手动流入阻塞进一步影响该阶段的血流动力学变量。因此，不建议将该方案用于评估再灌注前缺血期间的治疗方法。然而，其他实验设置，例如使用栓塞或结扎技术，可以与脊髓激光/多普勒针探头评估相结合，如本方案所述。

缺血/再灌注（SCI）引起的脊髓损伤是主动脉修复中最具破坏性的并发症之一，与结果减少1，2，3，4相关。目前SCI的预防和治疗方案包括大血流动力学参数的优化以及脑脊液压力（CSP）的正常化，以改善脊髓灌注压力2，5，6，7，8，9。尽管实施了这些操作，但SCI的发病率仍然在2%至31%之间，具体取决于主动脉修复的复杂程度10，11，12。
最近，微循环越来越受到关注13，14。微循环是细胞摄氧和代谢交换的区域，因此在器官功能和细胞完整性中起着关键作用13。微循环血流受损是与死亡率增加相关的组织缺血的主要决定因素15，16，17，18，19。脊髓微循环的损害与神经功能下降有关，结果20，21，22，23。因此，优化微灌注治疗SCI是最有希望的方法。尽管宏观循环优化，微循环干扰的持续存在，已经描述了26，27，28，29。这种血流动力学连贯性的丧失经常发生在包括缺血/再灌注在内的各种条件下，强调需要直接进行微循环评估和微循环靶向治疗26，27，30。
到目前为止，只有少数研究使用激光多普勒探针实时评估脊髓微循环行为20，31。现有研究经常使用微球注射技术，这些技术受到间歇使用和验尸分析的限制32，33。使用微球注射技术的不同测量次数受到不同波长微球的可用性的限制。此外，与激光多普勒技术相比，由于该方法需要死后组织处理和分析，因此无法实时评估微灌注。在这里，我们提出了一种实时评估猪缺血/再灌注大型动物模型中脊髓微循环的实验方案。
这项研究是一个大型动物项目的一部分，该项目结合了一项随机研究，比较了晶体与胶体对缺血/再灌注中微循环的影响，以及一项关于液体与血管加压药对脊髓微灌注的影响的探索性随机研究。流量探头2点校准以及压力尖端导管校准已在34中描述过。除了报道的方案外，荧光微球还用于测量脊髓微灌注，如前所述，使用每只动物的12个脊髓组织样本，样本1-6代表上脊髓，7-12代表下脊髓35，36。在完成激光多普勒记录和大血流动力学评估后，对每个测量步骤进行微球注入。如前所述，使用Kleinman评分进行组织病理学评估37。

<table><tbody><tr><td>CardioMed Flowmeter</td><td>Medistim AS, Oslo, Norway</td><td>CM4000</td><td>Flowmeter for Flow-Probe Femoral Artery</td></tr><tr><td>CardioMed Flow-Probe, 5mm</td><td>Medistim AS, Oslo, Norway</td><td>PS100051</td><td>Flow-Probe Femoral Artery</td></tr><tr><td>COnfidence probe,&amp;nbsp;</td><td>Transonic Systems Inc., Ithaca, NY, USA</td><td>MA16PAU</td><td>Flow-Probe Aorta</td></tr><tr><td>16 mm liners</td><td></td><td></td><td></td></tr><tr><td>DIVA Sevoflurane Vapor</td><td>Dr&amp;auml;ger Medical, L&amp;uuml;beck, Germany</td><td></td><td>Vapor</td></tr><tr><td>Hotline Level 1 Fluid Warmer</td><td>Smiths Medical Germany GmbH, Grasbrunn, Germany</td><td>HL-90-DE-230</td><td>Fluid Warmer</td></tr><tr><td>Infinity Delta</td><td>Dr&amp;auml;ger Medical, L&amp;uuml;beck, Germany</td><td></td><td>Basic Monitoring Hardware</td></tr><tr><td>Infinity Hemo</td><td>Dr&amp;auml;ger Medical, L&amp;uuml;beck, Germany</td><td></td><td>Basic Pressure Monitoring and Pulmonary Thermodilution Hardware</td></tr><tr><td>LabChart Pro</td><td>ADInstruments Ltd., Oxford, UK</td><td>v8.1.16</td><td>Synchronic Laser-Doppler, Blood Pressure, ECG and Blood-Flow Aquisition Software</td></tr><tr><td>LiquoGuard 7</td><td>M&amp;ouml;ller Medical GmbH, Fulda, Germany</td><td></td><td>Cerebrospinal Fluid Drainage System</td></tr><tr><td>Millar Micro-Tip Pressure Catheter (5F, Single, Curved, 120cm, PU/WD)</td><td>ADInstruments Ltd., Oxford, UK</td><td>SPR-350</td><td>Pressure-Tip Catheter Aorta</td></tr><tr><td>moor VMS LDF</td><td>moor Instruments, Devon, UK</td><td></td><td>Designated Laser-Doppler Hardware</td></tr><tr><td>moor VMS Research Software</td><td>moor Instruments, Devon, UK</td><td></td><td>Designated Laser-Doppler Software</td></tr><tr><td>Perivascular Flow Module</td><td>Transonic Systems Inc., Ithaca, NY, USA</td><td>TS 420</td><td>Flow-Module for Flow-Probe Aorta</td></tr><tr><td>PiCCO 2, Science Version</td><td>Getinge AB, G&amp;ouml;teborg, Sweden</td><td>v. 6.0</td><td>Blood Pressure and Transcardiopulmonary Monitoring Hard- and Software</td></tr><tr><td>PiCCO 5 Fr. 20cm</td><td>Getinge AB, G&amp;ouml;teborg, Sweden</td><td></td><td>Thermistor-tipped Arterial Line&amp;nbsp;</td></tr><tr><td>PowerLab</td><td>ADInstruments Ltd., Oxford, UK</td><td>PL 3516</td><td>Synchronic Laser-Doppler, Blood Pressure, ECG and Blood-Flow Aquisition Hardware</td></tr><tr><td>QuadBridgeAmp</td><td>ADInstruments Ltd., Oxford, UK</td><td>FE 224</td><td>Four Channel Bridge Amplifier for Laser-Doppler and Invasive Blood Pressure Aquisition</td></tr><tr><td>Silverline</td><td>Spiegelberg, Hamburg, Germany</td><td>ELD33.010.02</td><td>Cerebrospinal Fluid Drainage</td></tr><tr><td>SPSS statistical software package&amp;nbsp;</td><td>IBM SPSS Statistics Inc., Armonk, New York, USA</td><td>v. 27</td><td>Statistical Software</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Twinwarm Warming System</td><td>Moeck &amp;amp; Moeck GmbH, Hamburg, Germany</td><td>12TW921DE</td><td>Warming System</td></tr><tr><td>Universal II Warming Blanket</td><td>Moeck &amp;amp; Moeck GmbH, Hamburg, Germany</td><td>906</td><td>Warming Blanket</td></tr><tr><td>VP 3 Probe, 8mm length (individually manufactured)</td><td>moor Instruments, Devon, UK</td><td></td><td>Laser-Doppler Probe</td></tr><tr><td>Zeus</td><td>Dr&amp;auml;ger Medical, L&amp;uuml;beck, Germany</td><td></td><td>Anesthesia Machine</td></tr></tbody></table>

real-time assessment of spinal cord microperfusion in a porcine model of ischemia/reperfusion

脊髓损伤是主动脉修复的毁灭性并发症。尽管在预防和治疗脊髓损伤方面取得了进展，但其发病率仍然相当高，因此会影响患者的预后。微循环在组织灌注和氧气供应中起着关键作用，并且通常与大血流动力学分离。因此，脊髓微循环的直接评估对于开发微循环靶向疗法和评估有关脊髓微循环的现有方法至关重要。然而，大多数方法不能提供脊髓微循环的实时评估。本研究的目的是描述使用直接插入脊髓中的激光多普勒针探针进行实时脊髓微循环评估的标准化方案。我们使用缺血/再灌注的猪模型来诱导脊髓微循环的恶化。此外，还使用了荧光微球注射技术。最初，动物被麻醉和机械通气。此后，进行激光多普勒针探针插入，然后放置脑脊液引流。对下行主动脉的暴露进行正中胸骨切开术，以进行主动脉交叉钳夹。缺血/再灌注由乳糜泻上主动脉交叉钳夹术诱导，共48分钟，然后再灌注和血流动力学稳定。激光多普勒通量与大血流动力学评估同时进行。此外，使用自动脑脊液引流来维持稳定的脑脊压。完成方案后，处死动物，收获脊髓进行组织病理学和微球分析。该协议揭示了使用激光多普勒探针进行脊髓微灌注测量的可行性，并显示缺血期间以及再灌注后恢复期间显着减少。结果显示与荧光微球评估相当的行为。总之，这种新方案可能为未来在缺血/再灌注条件下使用实时脊髓微灌注评估的研究提供有用的大型动物模型。

所有六只动物都存活了下来，直到协议完成。动物体重为48.2±2.9公斤;五只动物是雄性的，一只动物是雌性。脊髓针探针插入以及脊髓通量测量在所有动物中都是可行的。
在主动脉交叉钳夹期间以及解疹和再灌注期间，实时脊髓微循环记录与脑微循环和大血流动力学记录相结合的示例如图3A，图3B所示。下降主动脉流的中断之后脊髓通量显着减少，而上升主动脉的压力增加（图3A）。再灌注导致相反的效果（图3B）。
宏观和微观循环参数的统计分析如 表1所示。混合模型估计的边际均值及其置信区间表明缺血期间脊髓通量显著降低。相反，缺血期间脑通量显着增加，如估计的边际平均值及其置信区间所示。这伴随着动脉压，心率和全身血管阻力的增加，而心输出量和每搏量减少。荧光微球分析显示，下脊髓的脊髓微循环血流量明显减少，而上脊髓没有显着变化，如估计的边际平均值及其置信区间所示。再灌注导致相反的效果。虽然在方案结束时心输出量，每搏量和动脉压进一步下降，但脊髓通量以及脊髓微循环血流量稳定。
这项研究的结果表明，激光/多普勒针探针能够检测脊髓微灌注的实时变化。正如预期的那样，缺血期间脊髓微循环的减少是急剧的，微循环通量最小。脊髓通量的恢复发生在再灌注后。用荧光微球评估的下脊髓灌注显示出可比的行为，因此支持该方法。不出所料，上脊髓灌注和脑通量表现出不同的行为。虽然脊髓微循环稳定，但在方案结束时，大循环下降，显示血流动力学相干性丧失。缺血期间降主动脉的血流量为零，但再灌注导致主动脉血流恢复。组织病理学分析显示脊髓轻度坏死，下脊髓评分在 0 到 2 之间，上脊髓评分在 0 到 1 之间。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62047/62047fig01.jpg"> 图1:激光/多普勒针探针在脊髓中的位置（A）椎体结构的手术暴露。（B） 使用静脉导管穿刺脊髓。（C）取下嵌针后插入针头探头。（D） 针头探头的固定。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62047/62047fig01large.jpg" target="_blank">请点击此处查看此图的放大版本。</a> 
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62047/62047fig02.jpg"> 图2:降主动脉的暴露以及流动探头和血管回路的放置。（A）在调动左肺的顶点并分裂隔膜的左外侧部分后，下行主动脉暴露。（B） 分割周围组织以进行手术暴露。（C） 在降主动脉周围放置一个支撑位，以确保适当的圆形曝光。（D） 在下降主动脉周围放置流动探头和容器环。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62047/62047fig02large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62047/62047fig03v2.jpg"> 图3:缺血和再灌注期间微循环和大血流动力学信号的样本记录。心电图的样本记录，使用微触头导管测量的升主动脉压力，使用超声流动探头测量的降主动脉中的流量，脊髓以及使用激光/多普勒针探头测量的脑微循环FLUX。（A） 50 s样本在缺血诱导期间通过乳糜泻主动脉交叉钳夹。（B）20 s样品在再灌注诱导期间通过轻轻地重新打开主动脉交叉钳。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62047/62047fig03largev2.jpg" target="_blank">请点击此处查看此图的放大版本。</a> 
<table border="1" fo:font-size="8px" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td colspan="2"></td>
			<td>M1型</td>
			<td>M2 型</td>
			<td>M3型</td>
			<td>M4型</td>
			<td>M5型</td>
		</tr><tr><td colspan="2">脊髓通量</td>
			<td>61.35 (41.96-89.70)</td>
			<td>6.78 (4.63-9.91)</td>
			<td>58.97 (40.33-86.22)</td>
			<td>66.05 (45.17-96.57)</td>
			<td>59.09 (40.41-86.40)</td>
		</tr><tr><td colspan="2">主效应测量点:p < 0.001</td>
			<td>成对比较 M1</td>
			<td>p < 0.001</td>
			<td>p = 0.878</td>
			<td>p = 0.777</td>
			<td>p = 0.886</td>
		</tr><tr><td colspan="2">脑通量</td>
			<td>41.12 (28.17-60.04)</td>
			<td>71.73 (49.13-104.73)</td>
			<td>60.34 (41.33-88.10)</td>
			<td>59.91 (36.93-78.71)</td>
			<td>49.82 (34.12-72.74)</td>
		</tr><tr><td colspan="2">主效应测量点:p = 0.023</td>
			<td>成对比较 M1</td>
			<td>p = 0.001</td>
			<td>p = 0.045</td>
			<td>p = 0.173</td>
			<td>p = 0.341</td>
		</tr><tr><td rowspan="2">脊髓微灌注（毫升/分钟/克）</td>
			<td rowspan="3">上脊髓</td>
			<td>0.071 (0.058-0.087)</td>
			<td>0.063 (0.052-0.078)</td>
			<td>0.088 (0.072-0.11)</td>
			<td>0.082 (0.067-0.100)</td>
			<td>0.083 (0.068-0.102)</td>
		</tr><tr><td rowspan="2">成对比较 M1</td>
			<td rowspan="2">p = 0.420</td>
			<td rowspan="2">p = 0.146</td>
			<td rowspan="2">p = 0.344</td>
			<td rowspan="2">p = 0.281</td>
		</tr><tr><td rowspan="2">主效应测量点:p < 0.001</td>
		</tr><tr><td rowspan="2">下脊髓</td>
			<td>0.079 (0.065-0.097)</td>
			<td>0.031 (0.026-0.039)</td>
			<td>0.111 (0.090-0.136)</td>
			<td>0.089 (0.073-0.110)</td>
			<td>0.105 (0.086-0.129)</td>
		</tr><tr><td>相互作用测量点 ·脊髓区:p < 0.001</td>
			<td>成对比较 M1</td>
			<td>p < 0.001</td>
			<td>p = 0.021</td>
			<td>p = 0.400</td>
			<td>p = 0.051</td>
		</tr><tr><td colspan="2">心输出量（升/分钟）</td>
			<td>4.15 (3.69-4.61)</td>
			<td>3.13 (2.67-3.60)</td>
			<td>3.30 (2.84-3.76)</td>
			<td>3.67 (3.20-4.13)</td>
			<td>2.67 (2.00-2.93)</td>
		</tr><tr><td colspan="2">主效应测量点:: p < 0.001</td>
			<td>成对比较 M1</td>
			<td>p < 0.001</td>
			<td>p = 0.007</td>
			<td>p = 0.125</td>
			<td>p < 0.001</td>
		</tr><tr><td colspan="2">心率（每分点）</td>
			<td>74.42 (53.70-95.15)</td>
			<td>131.09 (110.36-151.82)</td>
			<td>88.92 (68.19-109.65)</td>
			<td>80.62 (59.89-101.35)</td>
			<td>99.38 (78.65-120.11)</td>
		</tr><tr><td colspan="2">主效应测量点:p = 0.002</td>
			<td>成对比较 M1</td>
			<td>p < 0.001</td>
			<td>p = 0.314</td>
			<td>p = 0.666</td>
			<td>p = 0.092</td>
		</tr><tr><td colspan="2">冲程量（毫升）</td>
			<td>55.50 (49.20-61.81)</td>
			<td>25.33 (19.03-31.64)</td>
			<td>37.00 (30.69-43.31)</td>
			<td>45.33 (39.03-51.64)</td>
			<td>27.17 (20.86-33.47)</td>
		</tr><tr><td colspan="2">主效应测量点:p < 0.001</td>
			<td>成对比较 M1</td>
			<td>p < 0.001</td>
			<td>p < 0.001</td>
			<td>p = 0.004</td>
			<td>p < 0.001</td>
		</tr><tr><td colspan="2">收缩动脉压升主动脉 （mmHg）</td>
			<td>94.36 (85.20-103.52)</td>
			<td>122.05 (112.89-131.20)</td>
			<td>76.72 (67.56-85.88)</td>
			<td>88.36 (79.20-97.52)</td>
			<td>73.36 (64.20-82.52)</td>
		</tr><tr><td colspan="2">主效应测量点:p < 0.001</td>
			<td>成对比较 M1</td>
			<td>p < 0.001</td>
			<td>p = 0.006</td>
			<td>p = 0.321</td>
			<td>p = 0.002</td>
		</tr><tr><td colspan="2">升主动脉平均动脉压 （mmHg）</td>
			<td>78.18 (68.68-87.67)</td>
			<td>107.29 (97.80-116.78)</td>
			<td>59.08 (49.58-68.57)</td>
			<td>70.38 (60.89-79.87)</td>
			<td>58.35 (48.85-67.84)</td>
		</tr><tr><td colspan="2">主效应测量点:p < 0.001</td>
			<td>成对比较 M1</td>
			<td>p < 0.001</td>
			<td>p = 0.005</td>
			<td>p = 0.217</td>
			<td>p = 0.004</td>
		</tr><tr><td colspan="2">舒张动脉上行主动脉压 （mmHg）</td>
			<td>59.20 (49.41-69.00)</td>
			<td>93.76 (83.97-103.56)</td>
			<td>45.18 (35.38-54.98)</td>
			<td>52.48 (42.69-62.28)</td>
			<td>45.33 (35.54-55.13)</td>
		</tr><tr><td colspan="2">主效应测量点:p < 0.001</td>
			<td>成对比较 M1</td>
			<td>p < 0.001</td>
			<td>p = 0.038</td>
			<td>p = 0.302</td>
			<td>p = 0.040</td>
		</tr><tr><td colspan="2">全身血管阻力 （dyn x sec x cm-5）</td>
			<td>1421.13 (1236.94-1632.74)</td>
			<td>208089.94 (181128.10-239085.87)</td>
			<td>1335.36 (1162.29-1534.21)</td>
			<td>1412.62 (1229.54-1622.97)</td>
			<td>1807.46 (1573.21-2076.60)</td>
		</tr><tr><td colspan="2">主效应测量点:p < 0.001</td>
			<td>成对比较 M1</td>
			<td>p < 0.001</td>
			<td>p = 0.407</td>
			<td>p = 0.938</td>
			<td>p = 0.005</td>
		</tr><tr><td colspan="2">流量（升/分钟）下降主动脉</td>
			<td>3.27 (0.96-5.58)</td>
			<td rowspan="2">0</td>
			<td>3.27 (0.96-5.58)</td>
			<td>3.54 (1.23-5.85)</td>
			<td>4.54 (2.32-6.85)</td>
		</tr><tr><td colspan="2">主效应测量点:p = 0.003</td>
			<td>成对比较 M1</td>
			<td>p = 0.998</td>
			<td>p = 0.844</td>
			<td>p = 0.381</td>
		</tr></tbody></table>表1:方案期间血流动力学参数的变化。 这些值以基线调整的估计边际均值给出，置信区间为 95%。给出了每个参数测量点主效应的F检验的未调整p值以及上下脊髓微灌注区域与测量点之间的相互作用效应。还给出了单个测量点与M1成对比较的未调整p值。测量点为:M1 = 血流动力学优化前缺血/再灌注，M2 = 缺血期间，M3 = 再灌注后1小时 M4 = 缺血/再灌注后血流动力学优化，M5 = 4.5小时诱导缺血/再灌注后。

Watch this Scientific Journal Video about Real-Time Assessment of Spinal Cord Microperfusion in a Porcine Model of Ischemia/Reperfusion at JoVE.com

Real-Time Assessment of Spinal Cord Microperfusion in a Porcine Model of Ischemia/Reperfusion

脊髓微循环在脊髓损伤中起着关键作用。大多数方法不允许实时评估脊髓微循环，这对于开发微循环靶向疗法至关重要。在这里，我们提出了一种使用激光多普勒流针探针在缺血/再灌注的大型动物模型中的方案。

猪缺血/再灌注模型中脊髓微灌注的实时评估

Spinal cord injury is a devastating complication of aortic repair. Despite developments for the prevention and treatment of spinal cord injury, its ...

real-time-assessment-spinal-cord-microperfusion-porcine-model

Universidad San Sebastian

Research

JoVE Journal

Medicine

3.4K 次观看.  University Medical Center Hamburg-Eppendorf. 脊髓微循环在脊髓损伤中起着关键作用。大多数方法不允许实时评估脊髓微循环，这对于开发微循环靶向疗法至关重要。在这里，我们提出了一种使用激光多普勒流针探针在缺血/再灌注的大型动物模型中的方案。

视频: Real-Time Assessment of Spinal Cord Microperfusion in a Porcine Model of Ischemia/Reperfusion

Using Laser Doppler Imaging and Monitoring to Analyze Spinal Cord Microcirculation in Rat

Laser Doppler flowmetry (LDF) is a noninvasive method for blood flow (BF) measurement, which makes it preferable for measuring microcirculatory alterations of the spinal cord. In this article, our goal was to use both Laser Doppler imaging and monitoring to analyze the change of BF after spinal cord injury. Both the laser Doppler image scanner and the probe/monitor were being employed to obtain each readout. The data of LDPI provided a local distribution of BF, which gave an overview of perfusion around the injury site and made it accessible for comparative analysis of BF among different locations. By intensely measuring the probing area over a period of time, a combined probe was used to simultaneously measure the BF and oxygen saturation of the spinal cord, showing overall spinal cord perfusion and oxygen supply. LDF itself has a few limitations, such as relative flux, sensitivity to movement, and biological zero signal. However, the technology has been applied in clinical and experimental study due to its simple setup and rapid measurement of BF.

Here we present a combination of laser Doppler perfusion imaging (LDPI) and laser Doppler perfusion monitoring (LDPM) to measure spinal cord local blood flows and oxygen saturation (SO2), as well as a standardized procedure for introducing spinal cord trauma on rat.

激光多普勒成像与监测在大鼠脊髓微循环分析中的应用

Laser Doppler flowmetry (LDF) is a noninvasive method for blood flow (BF) measurement, which makes it preferable for measuring microcirculatory ...

科研

行为学

Reproducable Paraplegia by Thoracic Aortic Occlusion in a Murine Model of Spinal Cord Ischemia-reperfusion

Background 
Lower extremity paralysis continues to complicate aortic interventions. The lack of understanding of the underlying pathology has hindered advancements to decrease the occurrence this injury.&#160;The current model demonstrates reproducible lower&#160;extremity paralysis following thoracic aortic occlusion.
Methods 
Adult male C57BL6 mice were anesthetized with isoflurane. Through a cervicosternal incision the aorta was exposed. The descending thoracic aorta and left subclavian arteries were identified without entrance into pleural space. Skeletonization of these arteries was followed by immediate closure (Sham) or occlusion for 4 min (moderate ischemia) or 8 min (prolonged ischemia). The sternotomy and skin were closed and the mouse was transferred to warming bed for recovery.&#160; Following recovery, functional analysis was obtained at 12 hr intervals until 48 hr.
Results 
Mice that underwent sham surgery showed no observable hind&#160;limb deficit. Mice subjected to moderate ischemia for 4 min had minimal functional deficit at 12 hr followed by progression to complete paralysis at 48 hr. Mice subjected to prolonged ischemia had an immediate paralysis with no observable hind-limb movement at any point in the postoperative period. There was no observed intraoperative or post&#160;operative mortality.
Conclusion 
Reproducible lower extremity paralysis whether immediate or delayed can be achieved in a murine model. Additionally, by using a median sternotomy and careful dissection, high survival rates, and reproducibility can be achieved.

The lack of mechanistic understanding of spinal cord ischemia-reperfusion injury has hindered further adjuncts to prevent paraplegia following high risk aortic operations. Thus, the development of animal models is imperative. This manuscript demonstrates reproducible lower extremity paralysis following thoracic aortic occlusion in a murine model.

可重放截瘫胸主动脉阻断脊髓缺血再灌注的小鼠模型

Background
Lower extremity paralysis continues to complicate aortic interventions. The lack of understanding of the underlying pathology has hindered ...

医学

Contrast Enhanced Ultrasound Imaging for Assessment of Spinal Cord Blood Flow in Experimental Spinal Cord Injury

Reduced spinal cord blood flow (SCBF) (i.e., ischemia) plays a key role in traumatic spinal cord injury (SCI) pathophysiology and is accordingly an important target for neuroprotective therapies. Although several techniques have been described to assess SCBF, they all have significant limitations. To overcome the latter, we propose the use of real-time contrast enhanced ultrasound imaging (CEU). Here we describe the application of this technique in a rat contusion model of SCI. A jugular catheter is first implanted for the repeated injection of contrast agent, a sodium chloride solution of sulphur hexafluoride encapsulated microbubbles. The spine is then stabilized with a custom-made 3D-frame and the spinal cord dura mater is exposed by a laminectomy at ThIX-ThXII. The ultrasound probe is then positioned at the posterior aspect of the dura mater (coated with ultrasound gel). To assess baseline SCBF, a single intravenous injection (400 &#181;l) of contrast agent is applied to record its passage through the intact spinal cord microvasculature. A weight-drop device is subsequently used to generate a reproducible experimental contusion model of SCI. Contrast agent is re-injected 15 min following the injury to assess post-SCI SCBF changes. CEU allows for real time and in-vivo assessment of SCBF changes following SCI. In the uninjured animal, ultrasound imaging showed uneven blood flow along the intact spinal cord. Furthermore, 15 min post-SCI, there was critical ischemia at the level of the epicenter while SCBF remained preserved in the more remote intact areas. In the regions adjacent to the epicenter (both rostral and caudal), SCBF was significantly reduced. This corresponds to the previously described &#8220;ischemic penumbra zone&#8221;. This tool is of major interest for assessing the effects of therapies aimed at limiting ischemia and the resulting tissue necrosis subsequent to SCI.

Contrast Enhanced Ultrasound imaging is a reliable in-vivo tool for quantifying spinal cord blood flow in an experimental rat spinal cord injury model. This paper contains a comprehensive protocol for application of this technique in association with a contusion model of thoracic spinal cord injury.

超声造影成像脊髓血流量的评估实验脊髓损伤

Reduced spinal cord blood flow (SCBF) (i.e., ischemia) plays a key role in traumatic spinal cord injury (SCI) pathophysiology and is accordingly an ...

Improvement of a Closed Chest Porcine Myocardial Infarction Model by Standardization of Tissue and Blood Sampling Procedures

Myocardial ischemia reperfusion (I/R) injury contributes to almost half of the necrotic area after myocardial infarction. To date there is no approved drug to prevent or reduce myocardial I/R injury. The study and understanding of the pathophysiological mechanisms of myocardial I/R injury is essential to develop successful treatments. Large animal experiments are an important step in translational methods. The porcine model of acute myocardial infarction has been established and described by ourselves and others. We aimed to further improve the value of the model by focusing in detail on the sampling techniques for use in future experiments. Furthermore, we emphasize small but important steps that can affect the quality of the final results. To mimic the clinical situation of myocardial I/R injury, a percutaneous coronary intervention (PCI) catheter was inserted into the left anterior descending coronary artery (LAD) of an anesthetized pig. &#176;&#176;&#176;This model mimics acute myocardial infarction and PCI treatment in humans with the possibility of accurately determining the area at risk as well as the necrotic- and viable ischemic tissue. Here the model was used to investigate the effect of a bicyclic peptide inhibitor of FXIIa. The model can also be modified to allow longer reperfusion times to study later effects of myocardial infarction.

Here we demonstrate a protocol to standardize sampling procedures of an established porcine model of acute myocardial infarction in order to increase its translational value in the understanding of the pathophysiology of myocardial ischemia/reperfusion injury and to test novel drug candidates.

组织和采血程序标准化改善闭合性胸猪心肌梗死模型的研究

Myocardial ischemia reperfusion (I/R) injury contributes to almost half of the necrotic area after myocardial infarction. To date there is no approved ...

Intestinal Stem Cell Isolation and Culture in a Porcine Model of Segmental Small Intestinal Ischemia

Intestinal ischemia remains a major cause of morbidity and mortality in human and veterinary patients. Many disease processes result in intestinal ischemia, when the blood supply and therefore oxygen is decreased to the intestine. This leads to intestinal barrier loss and damage to the underlying tissue. Intestinal stem cells reside at the base of the crypts of Lieberk&#252;hn and are responsible for intestinal renewal during homeostasis and following injury. Ex vivo cell culture techniques have allowed for the successful study of epithelial stem cell interactions by establishing culture conditions that support the growth of three-dimensional epithelial organ-like systems (termed "enteroids" and "colonoids" from the small and large intestine, respectively). These enteroids are composed of crypt and villus-like domains and mature to contain all of the cell types found within the epithelium. Historically, murine models have been utilized to study intestinal injury. However, a porcine model offers several advantages including similarity of size as well as gastrointestinal anatomy and physiology to that of humans. By utilizing a porcine model, we establish a protocol in which segmental loops of intestinal ischemia can be created within a single animal, enabling the study of differing time points of ischemic injury and repair in vivo. Additionally, we describe a method to isolate and culture the intestinal stem cells from the ischemic loops of intestine, allowing for the continued study of epithelial repair, modulated by stem cells, ex vivo.

This protocol will enable readers to successfully establish a porcine model of segmental intestinal ischemia and subsequently isolate and culture intestinal stem cells for the study of epithelial repair following injury.

猪节段性小肠缺血模型中肠道干细胞的分离与培养

Intestinal ischemia remains a major cause of morbidity and mortality in human and veterinary patients. Many disease processes result in intestinal ...

Noninvasive and Invasive Renal Hypoxia Monitoring in a Porcine Model of Hemorrhagic Shock

Up to 50% of patients with trauma develop acute kidney injury (AKI), in part due to poor renal perfusion after severe blood loss. AKI is currently diagnosed based on a change in serum creatinine concentration from baseline or prolonged periods of decreased urine output. Unfortunately, baseline serum creatinine concentration data is unavailable in most patients with trauma, and current estimation methods are inaccurate. In addition, serum creatinine concentration may not change until 24-48 h after the injury. Lastly, oliguria must persist for a minimum of 6 h to diagnose AKI, making it impractical for early diagnosis. AKI diagnostic approaches available today are not useful for predicting risk during the resuscitation of patients with trauma. Studies suggest that urinary partial pressure of oxygen (PuO2) may be useful for assessing renal hypoxia. A monitor that connects the urinary catheter and the urine collection bag was developed to measure PuO2 noninvasively. The device incorporates an optical oxygen sensor that estimates PuO2 based on luminescence quenching principles. In addition, the device measures urinary flow and temperature, the latter to adjust for confounding effects of temperature changes. Urinary flow is measured to compensate for the effects of oxygen ingress during periods of low urine flow. This article describes a porcine model of hemorrhagic shock to study the relationship between noninvasive PuO2, renal hypoxia, and AKI development. A key element of the model is the ultrasound-guided surgical placement in the renal medulla of an oxygen probe, which is based on an unsheathed optical microfiber. PuO2 will also be measured in the bladder and compared to the kidney and noninvasive PuO2 measurements. This model can be used to test PuO2 as an early marker of AKI and assess PuO2 as a resuscitative endpoint after hemorrhage that is indicative of end-organ rather than systemic oxygenation.

Presented here is a protocol to measure renal oxygenation in the medulla and noninvasive urine oxygen partial pressure in a hemorrhagic shock porcine model to establish urine oxygen partial pressure as an early indicator of acute kidney injury (AKI) and a novel resuscitative endpoint.

猪出血性休克模型中的无创和侵入性肾缺氧监测

Up to 50% of patients with trauma develop acute kidney injury (AKI), in part due to poor renal perfusion after severe blood loss. AKI is currently ...

生物工程

Standardized Hemorrhagic Shock Induction Guided by Cerebral Oximetry and Extended Hemodynamic Monitoring in Pigs

Hemorrhagic shock ranks among the main reasons for severe injury-related death. The loss of circulatory volume and oxygen carriers can lead to an insufficient oxygen supply and irreversible organ failure. The brain exerts only limited compensation capacities and is particularly at high risk of severe hypoxic damage.This article demonstrates the reproducible induction of life-threatening hemorrhagic shock in a porcine model by means of calculated blood withdrawal. We titrate shock induction guided by near-infrared spectroscopy&#160;and extended hemodynamic monitoring to display systemic circulatory failure, as well as cerebral microcirculatory oxygen depletion. In comparison to similar models that primarily focus on predefined removal volumes for shock induction, this approach highlights a titration by means of the resulting failure of macro- and microcirculation.

Hemorrhagic shock is a severe complication in seriously injured patients, which leads to life-threatening oxygen undersupply. We present a standardized method to induce hemorrhagic shock via blood withdrawal in pigs that is guided by hemodynamics and microcirculatory cerebral oxygenation.

由脑氧测量和猪期扩展血液动力学监测指导的标准化出血性休克诱导

Hemorrhagic shock ranks among the main reasons for severe injury-related death. The loss of circulatory volume and oxygen carriers can lead to an ...

Post-Myocardial Infarction Heart Failure in Closed-chest Coronary Occlusion/Reperfusion Model in G&#246;ttingen Minipigs and Landrace Pigs

The development of heart failure is the most powerful predictor of long-term mortality in patients surviving acute myocardial infarction (MI). There is an unmet clinical need for prevention and therapy of post-myocardial infarction heart failure (post-MI HF). Clinically relevant pig models of post-MI HF are prerequisites for final proof-of-concept studies before entering into clinical trials in drug and medical device development.
Here we aimed to characterize a closed-chest porcine model of post-MI HF in adult G&#246;ttingen minipigs with long-term follow-up including serial cardiac magnetic resonance imaging (CMRI) and to compare it with the commonly used Landrace pig model.
MI was induced by intraluminal balloon occlusion of the left anterior descending coronary artery for 120 min in G&#246;ttingen minipigs and for 90 min in Landrace pigs, followed by reperfusion. CMRI was performed to assess cardiac morphology and function at baseline in both breeds and at 3 and 6 months in G&#246;ttingen minipigs and at 2 months in Landrace pigs, respectively.
Scar sizes were comparable in the two breeds, but MI resulted in a significant decrease of left ventricular ejection fraction (LVEF) only in G&#246;ttingen minipigs, while Landrace pigs did not show a reduction of LVEF. Right ventricular (RV) ejection fraction increased in both breeds despite the negligible RV scar sizes. In contrast to the significant increase of left ventricular end-diastolic (LVED) mass in Landrace pigs at 2 months, G&#246;ttingen minipigs showed a slight increase in LVED mass only at 6 months.
In summary, this is the first characterization of post-MI HF in G&#246;ttingen minipigs in comparison to Landrace pigs, showing that the G&#246;ttingen minipig model reflects post-MI HF parameters comparable to the human pathology. We conclude that the G&#246;ttingen minipig model is superior to the Landrace pig model to study the development of post-MI HF.

The overall goal of the current study is to present the techniques of induction of myocardial infarction (MI) and post-myocardial infarction heart failure (post-MI HF) in closed-chest, adult G&#246;ttingen minipigs and the characterization of post-MI HF model in G&#246;ttingen minipigs as compared to Landrace pigs.

封闭胸冠状动脉闭塞/输液模型中的心肌梗塞后心力衰竭

The development of heart failure is the most powerful predictor of long-term mortality in patients surviving acute myocardial infarction (MI). There is an ...

Development and Validation of a Porcine Ex Situ Isolated Liver Perfusion Model 

Porcine Normothermic Isolated Liver Perfusion

Porcine models of liver ex situ normothermic machine perfusion (NMP) are increasingly being used in transplant research. Contrary to rodents, porcine livers are anatomically and physiologically close to humans, with similar organ size and bile composition. NMP preserves the liver graft at near-to-physiological conditions by recirculating a warm, oxygenated, and nutrient-enriched red blood cell-based perfusate through the liver vasculature. NMP can be used to study ischemia-reperfusion injury, preserve a liver ex situ before transplantation, assess the liver's function prior to implantation, and provide a platform for organ repair and regeneration. Alternatively, NMP with a whole blood-based perfusate can be used to mimic transplantation. Nevertheless, this model is labor-intensive, technically challenging, and carries a high financial cost.
In this porcine NMP model, we use warm ischemic damaged livers (corresponding to donation after circulatory death). First, general anesthesia with mechanical ventilation is initiated, followed by the induction of warm ischemia by clamping the thoracic aorta for 60 min. Cannulas inserted in the abdominal aorta and portal vein allow flush-out of the liver with cold preservation solution. The flushed-out blood is washed with a cell saver to obtain concentrated red blood cells. Following hepatectomy, cannulas are inserted in the portal vein, hepatic artery, and infra-hepatic vena cava and connected to a closed perfusion circuit primed with a plasma expander and red blood cells. A hollow fiber oxygenator is included in the circuit and coupled to a heat exchanger to maintain a pO2 of 70-100 mmHg at 38 &#176;C. NMP is achieved by a continuous flow directly through the artery and via a venous reservoir through the portal vein. Flows, pressures, and blood gas values are continuously monitored. To evaluate the liver injury, perfusate and tissue are sampled at predefined time points; bile is collected via a cannula in the common bile duct.

The porcine model of liver normothermic machine perfusion (NMP), described here, can be successfully used to study NMP as a preservation strategy, a tool for viability assessment, and a platform for organ repair. It holds a high translational value, however it is technically challenging and labor-intensive.

Porcine models of liver ex situ normothermic machine perfusion (NMP) are increasingly being used in transplant research. Contrary to rodents, porcine ...

Laser Doppler Needle Probe Insertion: A Technique for Real-time Quantification of Blood Flow within the Spinal Cord in a Porcine Model

Source: Behem, C. R. et al., <a href="https://www-jove-com.bdigitaluss.remotexs.co/v/62047">Real-Time Assessment of Spinal Cord Microperfusion in a Porcine Model of Ischemia/Reperfusion.</a> J. Vis. Exp. (2020).
This video demonstrates the placement of a laser Doppler needle probe in the spinal cord of a porcine model. This helps in the continuous quantification of blood supply to the spinal cord, an important assessment in spinal cord injury.

激光多普勒针探针插入:一种在猪模型中实时量化脊髓内血流的技术

Source: Behem, C. R. et al., Real-Time Assessment of Spinal Cord Microperfusion in a Porcine Model of Ischemia/Reperfusion. J. Vis. Exp. (2020).
This ...