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Medizin

猪缺血/再灌注模型中脊髓微灌注的实时评估

Published: December 10, 2020
doi:
10.3791/62047
, , , , , , , , , , ,
1Department of Anesthesiology, Center of Anesthesiology and Intensive Care Medicine,University Medical Center Hamburg-Eppendorf, 2University Department for Vascular Surgery and Department of Operative Medicine,Medical University of Innsbruck, 3Department of Medical Biometry and Epidemiology,University Medical Center Hamburg-Eppendorf, 4Department of Vascular Medicine,University Heart and Vascular Center Hamburg (UHZ), 5Department of Cardiology,Rostock University Medical Center, 6University Department for Cardiac Surgery,Heart Center Leipzig

Summary

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。

Abstract

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。

Introduction

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。

Protocol

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?…

Representative Results

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…

Discussion

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/…

Offenlegungen

The authors have nothing to disclose.

Acknowledgements

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。

Materials

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

Spinal CordMicroperfusionIschemia/reperfusionReal-time AssessmentPorcine ModelLaser DopplerCerebrospinal Fluid PressureMacrohemodynamicsMinimally InvasiveAnesthesiologySurgical Procedure

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Behem, C. R., Friedheim, T., Wipper, S. H., Pinnschmidt, H. O., Graessler, M. F., Gaeth, C., Holthusen, H., Rapp, A., Suntrop, T., Haunschild, J., Etz, C. D., Trepte, C. J. C. Real-Time Assessment of Spinal Cord Microperfusion in a Porcine Model of Ischemia/Reperfusion. J. Vis. Exp. (166), e62047, doi:10.3791/62047 (2020).
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