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/...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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		<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,&#160;</td>
			<td rowspan="2">Transonic Systems Inc., Ithaca, NY, USA</td>
			<td rowspan="2">MA16PAU</td>
			<td rowspan="2">Flow-Probe Aorta</td>
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		<tr>
			<td>16 mm liners</td>
		</tr>
		<tr>
			<td>DIVA Sevoflurane Vapor</td>
			<td>Dr&#228;ger Medical, L&#252;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&#228;ger Medical, L&#252;beck, Germany</td>
			<td></td>
			<td>Basic Monitoring Hardware</td>
		</tr>
		<tr>
			<td>Infinity Hemo</td>
			<td>Dr&#228;ger Medical, L&#252;beck, Germany</td>
			<td></td>
			<td>Basic Pressure Monitoring and Pulmonary Thermodilution Hardware</td>
		</tr>
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			<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&#246;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&#246;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&#246;teborg, Sweden</td>
			<td></td>
			<td>Thermistor-tipped Arterial Line&#160;</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 rowspan="2">SPSS statistical software package&#160;</td>
			<td rowspan="2">IBM SPSS Statistics Inc., Armonk, New York, USA</td>
			<td rowspan="2">v. 27</td>
			<td rowspan="2">Statistical Software</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td>Twinwarm Warming System</td>
			<td>Moeck &#38; Moeck GmbH, Hamburg, Germany</td>
			<td>12TW921DE</td>
			<td>Warming System</td>
		</tr>
		<tr>
			<td>Universal II Warming Blanket</td>
			<td>Moeck &#38; 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&#228;ger Medical, L&#252;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，<strong class="xf...

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

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

Normothermic Cardiac Arrest and Cardiopulmonary Resuscitation: A Mouse Model of Ischemia-Reperfusion Injury

Acute Kidney Injury (AKI) is a common, highly lethal, complication of critical illness which has a high mortality1-4 and which is most 
frequently caused by whole-body hypoperfusion.5,6 Successful reproduction of whole-body hypoperfusion in rodent models has been fraught with 
difficulty.7-9,9,10 Models which employ focal ischemia have repeatedly demonstrated results which do not translate to the clinical setting, and larger 
animal models which allow for whole body hypoperfusion lack access to the full toolset of genetic manipulation possible in the mouse.11,12 However, in recent 
years a mouse model of cardiac arrest and cardiopulmonary resuscitation has emerged which can be adapted to model AKI.13 This model reliably reproduces 
physiologic, functional, anatomic, and histologic outcomes seen in clinical AKI, is rapidly repeatable, and offers all of the significant advantages of a murine surgical 
model, including access to genetic manipulative techniques, low cost relative to large animals, and ease of use. Our group has developed extensive experience 
with use of this model to assess a number of organ-specific outcomes in AKI.14,15

A powerful model for perioperative and critical care related acute kidney injury is presented. Using whole body hypoperfusion induced by cardiac arrest it is possible to nearly replicate the histologic and functional changes of clinical AKI.

常温心脏骤停和心肺复苏术：缺血再灌注损伤的小鼠模型

Acute Kidney Injury (AKI) is a common, highly lethal, complication of critical illness which has a high mortality1-4 and which is most 
frequently caused ...

科研

医学

Acute and Chronic Tactile Sensory Testing after Spinal Cord Injury in Rats

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory testing in rat SCI models is needed. However, until recently, no single testing approach had been validated for SCI so that standardized methods have not been implemented across labs. Additionally, available testing methods could not be implemented acutely or when severe motor impairments existed, preventing studies of the development of SCI-induced allodynia3. Here we present two validated sensory testing methods using von Frey Hair (VFH) monofilaments which quantify changes in tactile sensory thresholds after SCI4-5. One test is the well-established Up-Down test which demonstrates high sensitivity and specificity across different SCI severities when tested chronically5. The other test is a newly-developed dorsal VFH test that can be applied acutely after SCI when allodynia develops, prior to motor recovery4-5. Each VFH monofilament applies a calibrated force when touched to the skin of the hind paw until it bends. In the up-down method, alternating VFHs of higher or lower forces are used on the plantar L5 dermatome to delineate flexor withdrawal thresholds. Successively higher forces are applied until withdrawal occurs then lower force VFHs are used until withdrawal ceases. The tactile threshold reflects the force required to elicit withdrawal in 50% of the stimuli. For the new test, each VFH is applied to the dorsal L5 dermatome of the paw while the rat is supported by the examiner. The VFH stimulation occurs in ascending order of force until at least 2 of 3 applications at a given force produces paw withdrawal. Tactile sensory threshold is the lowest force to elicit withdrawal 66% of the time. Acclimation, testing and scoring procedures are described. Aberrant trials that require a retest and typical trials are defined. Animal use was approved by Ohio State University Animal Care and Use Committee.

We describe two tactile sensory testing methods for acute or chronic periods of spinal cord injury in rats. These validated procedures can detect the development and maintenance of allodynia-like sensations.

在大鼠脊髓损伤后的急性和慢性触觉测试

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory ...

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury (IRI) of the Rat Cremaster Muscle

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, intestinal ischemia as well as following transplant and cardiovascular surgery.1 Reperfusion of previously ischemic tissue, while essential for the prevention of irreversible tissue injury, elicits excessive inflammation of the affected tissue. Adjacent to the production of reactive oxygen species, activation of the complement system and increased microvascular permeability, the activation of leukocytes is one of the principle actors in the pathological cascade of inflammatory tissue damage during reperfusion.2, 3 Leukocyte activation is a multistep process consisting of rolling, firm adhesion and transmigration and is mediated by a complex interaction between adhesion molecules in response to chemoattractants such as complement factors, chemokines, or platelet-activating factor.4
While leukocyte rolling in postcapillary venules is predominantly mediated by the interaction of selectins5 with their counter ligands, firm adhesion of leukocytes to the endothelium is selectin-controlled via binding to intercellular adhesion molecules (ICAM) and vascular cellular adhesion molecules (VCAM).6, 7 
Gold standard for the in vivo observation of leukocyte-endothelial interaction is the technique of intravital microscopy, first described in 1968.8
Though various models of IRI (ischemia-reperfusion injury) have been described for various organs, 9-12 only few are suitable for direct visualization of leukocyte recruitment in the microvascular bed on a high level of image quality.8 
We here promote the digital intravital epifluorescence microscopy of the postcapillary venule in the cremasteric microcirculation of the rat 13 as a convenient method to qualitatively and quantitatively analyze leukocyte recruitment for IRI-research in striated muscle tissue and provide a detailed manual for accomplishing the technique. We further illustrate common pitfalls and provide useful tips which should enable the reader to truly appreciate, and safely perform the method.
In a step by step protocol we depict how to get started with respiration controlled anesthesia under sufficient monitoring to keep the animal firmly anesthetized for longer periods of time. We then describe the cremasteric preparation as a thin flat sheet for outstanding optical resolution and provide a protocol for leukocyte imaging in IRI that has been well established in our laboratories.

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury IRI of the Rat Cremaster Muscle

Digital intravital epifluorescence microscopy of postcapillary venules in the cremasteric microcirculation is a convenient method to gain insights into leukocyte-endothelial interaction in vivo in ischemia-reperfusion injury (IRI) of striated muscle tissue. We here provide a detailed protocol to safely perform the technique and discuss its applications and limitations.

实时数字成像大鼠提睾肌缺血再灌注损伤（IRI）的白细胞 - 内皮细胞相互作用

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, ...

A Contusion Model of Severe Spinal Cord Injury in Rats

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is described to obtain a consistent model of severe SCI. Use of a stereotactic frame and computer controlled impactor allows for creation of reproducible injury. Hypothermia and urinary tract infection pose significant challenges in the post-operative period. Careful monitoring of animals with daily weight recording and bladder expression allows for early detection of post-operative complications. The functional results of this contusion model are equivalent to transection models. The contusion model can be utilized to evaluate the efficacy of both neuroprotective and neuroregenerative approaches.

A contusion model of severe spinal cord injury is described. Detailed pre-operative, operative and post-operative steps are described to obtain a consistent model.

挫伤模型脊髓严重损伤的保护作用

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is ...

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 ...

Controlled Cortical Impact Model for Traumatic Brain Injury

Every year over a million Americans suffer a traumatic brain injury (TBI). Combined with the incidence of TBIs worldwide, the physical, emotional, social, and economical effects are staggering. Therefore, further research into the effects of TBI and effective treatments is necessary. The controlled cortical impact (CCI) model induces traumatic brain injuries ranging from mild to severe. This method uses a rigid impactor to deliver mechanical energy to an intact dura exposed following a craniectomy. Impact is made under precise parameters at a set velocity to achieve a pre-determined deformation depth. Although other TBI models, such as weight drop and fluid percussion, exist, CCI is more accurate, easier to control, and most importantly, produces traumatic brain injuries similar to those seen in humans. However, no TBI model is currently able to reproduce pathological changes identical to those seen in human patients. The CCI model allows investigation into the short-term and long-term effects of TBI, such as neuronal death, memory deficits, and cerebral edema, as well as potential therapeutic treatments for TBI.

Traumatic brain injuries (TBIs) remain a serious health problem. Using the controlled cortical impact surgery model, research on the effects of TBI and possible treatment methods may be performed.

控制皮层影响模型创伤性脑损伤

Every year over a million Americans suffer a traumatic brain injury (TBI).  Combined with the incidence of TBIs worldwide, the physical, emotional, ...

A Radio-telemetric System to Monitor Cardiovascular Function in Rats with Spinal Cord Transection and Embryonic Neural Stem Cell Grafts

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter connected to a radio transmitter into the femoral artery of rats that underwent a T4 spinal cord transection with or without grafting of embryonic brainstem-derived neural stem cells expressing green fluorescent protein. Compared to other methods such as cannula insertion or tail-cuff, telemetry is advantageous to continuously monitor blood pressure and heart rate in freely moving animals. It is also capable of long term multiple data acquisitions. In spinal cord injured rats, basal cardiovascular data under unrestrained condition and autonomic dysreflexia in response to colorectal distension were successfully recorded. In addition, cardiovascular parameters before and after SCI can be compared in the same rat if a transmitter is implanted before a spinal cord transection. One limitation of the described telemetry procedure is that implantation in the femoral artery may influence the blood supply to the ipsilateral hindlimb.

We present a protocol for using a radio-telemetric system to record cardiovascular parameters in T4 spinal cord transected rats eight weeks after embryonic brainstem neural stem cell grafting into the lesion site. Telemetry is an advanced technique to accurately evaluate cardiovascular function in conscious freely moving spinal cord injured rats.

无线电遥测系统，以监测大鼠脊髓横断和胚胎神经干细胞移植在心血管功能

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter ...

Calibrated Forceps Model of Spinal Cord Compression Injury

Compression injuries of the murine spinal cord are valuable animal models for the study of spinal cord injury (SCI) and spinal regenerative therapy. The calibrated forceps model of compression injury is a convenient, low cost, and very reproducible animal model for SCI. We used a pair of modified forceps in accordance with the method published by Plemel et al. (2008) to laterally compress the spinal cord to a distance of 0.35 mm. In this video, we will demonstrate a dorsal laminectomy to expose the spinal cord, followed by compression of the spinal cord with the modified forceps. In the video, we will also address issues related to the care of paraplegic laboratory animals. This injury model produces mice that exhibit impairment in sensation, as well as impaired hindlimb locomotor function. Furthermore, this method of injury produces consistent aberrations in the pathology of the SCI, as determined by immunohistochemical methods. After watching this video, viewers should be able to determine the necessary supplies and methods for producing SCI of various severities in the mouse for studies on SCI and/or treatments designed to mitigate impairment after injury.

Spinal cord injury models should be highly reproducible. We demonstrate that the calibrated forceps compression model of spinal cord injury is an easy to use surgical method for generating reproducible injuries to the murine spinal cord.

脊髓压迫损伤的校准钳型

Compression injuries of the murine spinal cord are valuable animal models for the study of spinal cord injury (SCI) and spinal regenerative therapy. The ...

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 ...

Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for myocardial infarction and cardiac complications of thoracic surgery. The ability of certain drugs to precondition the myocardium against ischemia reperfusion injury has led to multiple clinical trials, with little success. The isolated heart model allows acute observation of the functional effects of ischemia reperfusion injury in real time, including the effects of various pharmacological interventions administered at any time-point before or within the ischemia-reperfusion injury window. Since brief periods of ischemia can precondition the heart against ischemic injury, in situ aortic cannulation is performed to allow for functional assessment of non-preconditioned myocardium. A saline filled balloon is placed into the left ventricle to allow for real-time measurement of pressure generation. Ischemic injury is simulated by the cessation of perfusion buffer flow, followed by reperfusion. The duration of both ischemia and reperfusion can be modulated to examine biochemical events at any given time-point. Although the Langendorff isolated heart model does not allow for the consideration of systemic events affecting ischemia and reperfusion, it is an excellent model for the examination of acute functional and biochemical events within the window of ischemia reperfusion injury as well as the effect of pharmacological intervention on cardiac pre- and postconditioning. The goal of this protocol is to demonstrate how to perform in situ aortic cannulation and heart excision followed by ischemia/reperfusion injury in the Langendorff model.

The isolated rat heart is an enduring model for ischemia reperfusion injury. Here, we describe the process of harvesting the beating heart from a rat via in situ aortic cannulation, Langendorff perfusion of the heart, simulated ischemia-reperfusion injury, and infarct staining to confirm the extent of ischemic insult.

诱导和缺血再灌注损伤评估中的Langendorff灌注鼠心

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for ...