高频颅内双体超声波的苏巴拉赫诺德出血的穆林模型脑血管痉挛分析
1Department of Neurosurgery,University Medical Center of the Johannes Gutenberg-University of Mainz, 2Center for Cardiology – Cardiology I,University Medical Center of the Johannes Gutenberg-University of Mainz, 3Center for Thrombosis and Hemostasis (CTH),University Medical Center of the Johannes Gutenberg-University of Mainz, 4German Center for Cardiovascular Research (DZHK) – Partner site Rhine-Main, 5Department of Anesthesiology,University Medical Center of the Johannes Gutenberg-University of Mainz
Summary
本手稿的目的是提出一种基于声学的方法，允许 在体内 成像小鼠脑动脉的血流。我们演示了它的应用，以确定与血管痉挛相关的血流速度的变化，在亚拉氏出血（SAH）的穆林模型。
Abstract
脑血管痉挛发生在亚拉氏出血后的几周内，这是一种出血性中风，导致脑缺血延迟。在使用SAH的murine模型的实验研究中遇到的一个问题是缺乏对小鼠脑血管痉挛进行 体内 监测的方法。在这里，我们演示了高频超声波在小鼠身上进行颅面双子全息检查的应用。使用该方法，可以识别内部胡萝卜动脉 （ICA）。吸入SAH后，颅内ICA的血流速度显著加快，而颅内ICA的血流速度仍然很低，表明脑血管痉挛。最后，这里演示的方法允许在murine SAH模型中对脑血管痉挛进行功能性、非侵入性的 体内 监测。
Introduction
自发性亚拉氏出血（SAH）是一种出血性中风，主要由颅内动脉瘤1破裂引起。神经学结果主要受两个因素的影响：早期脑损伤（EBI），这是由出血和相关的瞬态全球脑缺血的影响，和延迟脑缺血（DCI），这发生在出血后2，3的几个星期。据报道，DCI影响高达30%的SAH患者2。DCI的病理生理学涉及血管造影脑血管痉挛，一种由微血管痉挛和微血栓引起的干扰微循环，皮质扩散性抑郁症，以及炎症4引起的影响。不幸的是，确切的病理生理学仍然不清楚，没有可用的治疗方法，有效地防止DCI3。因此，DCI在许多临床和实验研究中都进行了研究。
如今，大多数关于SAH的实验研究都使用小型动物模型，特别是在小鼠5、6、7、8、9、10、11、12、13。在此类研究中，脑血管痉挛经常被调查为终点。这是常见的，以确定血管痉挛前活体的程度。这是因为脑血管痉挛的体内检查的非侵入性方法需要很短的麻醉时间，只对动物造成很少的困扰。然而，在体内检查脑血管痉挛将是有利的。这是因为它允许在体内对小鼠血管痉挛的研究（即在SAH诱导后的几天内在不同时间点对脑血管痉挛进行成像）。这将提高在不同时间点获得的数据的可比性。此外，使用纵向研究设计是减少动物数量的策略。
在这里，我们演示使用高频颅外超声波来确定小鼠脑动脉的血流量。我们表明，类似于颅内多普勒声像（TCD）或颅内颜色编码的双体声学（TCCD）在临床实践14，15，16，17，18，这种方法可用于监测脑血管痉挛，测量血流速度后，SAH诱导在Murine模型。
Protocol
动物实验由负责动物护理委员会（莱茵兰-普法尔茨）批准，并根据德国动物福利法（TierSchG）进行。遵循所有适用的国际、国家和机构关于照顾和使用动物的准则。在这项研究中，我们进行了女性C57BL/6N小鼠颅内和颅外动脉血流速度的测量，年龄在11-12周，体重在19-21克之间。这些老鼠要么接受SAH诱导手术，要么接受虚假手术，这在其他地方已经详细描述了10、12、13。<sup …
Representative Results
在6只小鼠中，其中3只小鼠使用血管内丝穿孔模型诱导SAH，3只获得假手术，颅内胡萝卜动脉（ICA）和颅外ICA的血流速度在手术前一天确定，手术后1天、3天和7天确定。测量是作为另一项研究的回声心动图检查的一部分，麻醉下与异氟兰，同时保持体温在37°C19。 手术前，颅外和颅内血流速度，以及颅内和颅外血流的商数在SAH和假动物之间相似。在SAH上岗后的?…
Discussion
据我们所知，这项研究是第一次提出一个协议，以监测大脑血管痉挛在SAH的murine模型与高频颅面颜色编码双面超声波。我们表明，这种方法可以测量小鼠SAH诱导后颅内血流速度的增加。在人类医学中，这种现象是众所周知的3，15。多项临床研究表明，大颅内动脉血流速度升高和颅内和颅内血流速度升高是血管变窄的功能后果，与血管血管痉挛相关<sup cla…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者要感谢斯特凡·金德尔为视频中的插图所做的准备。PW、MM 和 SHK 得到了德国联邦教育和研究部 （BMBF 01EO1503） 的支持。这项工作得到了德国研究基金会（DFG INST 371/47-1 FUGG）的一笔大型仪器赠款的支持。MM得到了埃尔斯·克雷纳-弗雷塞纽斯-斯蒂夫通（2020_EKEA.144）的赠款的支持。
Materials
| Balea hair removal creme | Balea; Germany | ASIN B0759XM39V | hair removal creme |
| C57BL/6N mice | Janvier; Saint-Berthevin Cedex, France | n.a. | mice |
| Corneregel | Bausch&Lomb; Rochester, NY, USA | REF 81552983 | eye ointment, lube |
| cotton swabs | Hecht Assistent; Sondenheim vor der Röhn, Germany | REF 44302010 | cotton swabs |
| Ecco-XS razor | Tondeo; Soligen, Germany | DE 28693396 | razor |
| Electrode cream | GE; Boston, MA, USA | REF 21708318 | conductive paste |
| Heating plate | Medax; Kiel, Germany | 2005-205-01 | |
| Isoflurane | Abvie; Wiesbaden, Germany | n.a. | volatile anesthetic |
| Leukofix | BSN medical; Hamburg, Germany | REF 02137-00 | tape |
| Mechanical arm + micromanipulator | VisualSonics; FujiFilm, Toronto, CA | P/N 11277 | |
| Microbac tissues | Paul Hartmann AG; Hamburg, Germany | REF 981387 | antimicrobial tissues |
| MZ400, 38 MHz linear array transducer | VisualSonics; FujiFilm, Toronto, CA | REF 51068-30 | ultrasound transducer |
| Sonosid | ASID Bonz GmbH; Herrenberg, Germany | REF 782010 | ultrasonography gel |
| Ultrasound platform with heating plate and ECG-recording | VisualSonics; FujiFilm, Toronto, CA | P/N 11179 | |
| UniVet-Porta | Groppler; Oberperasberg, Germany | S/N BKGM0437 | isoflurane vaporizer |
| Vevo3100 | VisualSonics; FujiFilm, Toronto, CA | REF 51073-45 | ultrasonography device |
| VevoLab software | VisualSonics; FujiFilm, Toronto, CA | n.a. | evaluation software |
References
- Macdonald, R. L., Schweizer, T. A. Spontaneous subarachnoid haemorrhage. Lancet. 389 (10069), 655-666 (2017).
- Macdonald, R. L. Delayed neurological deterioration after subarachnoid haemorrhage. Nature Reviews Neurology. 10 (1), 44-58 (2014).
- Francoeur, C. L., Mayer, S. A. Management of delayed cerebral ischemia after subarachnoid hemorrhage. Critical Care. 20 (1), 277 (2016).
- van Lieshout, J. H., et al. An introduction to the pathophysiology of aneurysmal subarachnoid hemorrhage. Neurosurgical Review. , (2017).
- Altay, T., et al. A novel method for subarachnoid hemorrhage to induce vasospasm in mice. J Neurosci Methods. 183 (2), 136-140 (2009).
- Momin, E. N., et al. Controlled delivery of nitric oxide inhibits leukocyte migration and prevents vasospasm in haptoglobin 2-2 mice after subarachnoid hemorrhage. Neurosurgery. 65 (5), 937-945 (2009).
- Froehler, M. T., et al. Vasospasm after subarachnoid hemorrhage in haptoglobin 2-2 mice can be prevented with a glutathione peroxidase mimetic. Journal of Clinical Neuroscience. 17 (9), 1169-1172 (2010).
- Provencio, J. J., Altay, T., Smithason, S., Moore, S. K., Ransohoff, R. M. Depletion of Ly6G/C(+) cells ameliorates delayed cerebral vasospasm in subarachnoid hemorrhage. Journal of Neuroimmunology. 232 (1-2), 94-100 (2011).
- Kamp, M. A., et al. Evaluation of a murine single-blood-injection SAH model. PLoS One. 9 (12), 114946 (2014).
- Luh, C., et al. The Contractile Apparatus Is Essential for the Integrity of the Blood-Brain Barrier After Experimental Subarachnoid Hemorrhage. Translational Stroke Research. , (2018).
- Neulen, A., et al. A Volumetric Method for Quantification of Cerebral Vasospasm in a Murine Model of Subarachnoid Hemorrhage. Journal of Visualized Experiments. (137), (2018).
- Neulen, A., et al. Large Vessel Vasospasm Is Not Associated with Cerebral Cortical Hypoperfusion in a Murine Model of Subarachnoid Hemorrhage. Translational Stroke Research. , (2018).
- Neulen, A., et al. Neutrophils mediate early cerebral cortical hypoperfusion in a murine model of subarachnoid haemorrhage. Scientific Reports. 9 (1), 8460 (2019).
- Neulen, A., et al. Volumetric analysis of intracranial vessels: a novel tool for evaluation of cerebral vasospasm. Int J Comput Assist Radiol Surg. 14 (1), 157-167 (2019).
- Washington, C. W., Zipfel, G. J. Participants in the International Multi-disciplinary Consensus Conference on the Critical Care Management of Subarachnoid, H. Detection and monitoring of vasospasm and delayed cerebral ischemia: a review and assessment of the literature. NeuroCritical Care. 15 (2), 312-317 (2011).
- Greke, C., et al. Image-guided transcranial Doppler sonography for monitoring of defined segments of intracranial arteries. Journal of Neurosurgical Anesthesiology. 25 (1), 55-61 (2013).
- Neulen, A., Prokesch, E., Stein, M., Konig, J., Giese, A. Image-guided transcranial Doppler sonography for monitoring of vasospasm after subarachnoid hemorrhage. Clinical Neurology and Neurosurgery. 145, 14-18 (2016).
- Neulen, A., et al. Image-Guided Transcranial Doppler Ultrasound for Monitoring Posthemorrhagic Vasospasms of Infratentorial Arteries: A Feasibility Study. World Neurosurgery. 134, 284-291 (2020).
- Neulen, A., et al. Correlation of cardiac function and cerebral perfusion in a murine model of subarachnoid hemorrhage. Scientific Reports. 11 (1), 3317 (2021).
- Neulen, A., et al. A segmentation-based volumetric approach to localize and quantify cerebral vasospasm based on tomographic imaging data. PLoS One. 12 (2), 0172010 (2017).
- Marbacher, S., et al. Systematic Review of In Vivo Animal Models of Subarachnoid Hemorrhage: Species, Standard Parameters, and Outcomes. Translational Stroke Research. , (2018).
- Figueiredo, G., et al. Comparison of digital subtraction angiography, micro-computed tomography angiography and magnetic resonance angiography in the assessment of the cerebrovascular system in live mice. Clinical Neuroradiology. 22 (1), 21-28 (2012).
- Lindegaard, K. F., Nornes, H., Bakke, S. J., Sorteberg, W., Nakstad, P. Cerebral vasospasm diagnosis by means of angiography and blood velocity measurements. Acta Neurochirurgica. 100 (1-2), 12-24 (1989).
- Cassia, G. S., Faingold, R., Bernard, C., Sant’Anna, G. M. Neonatal hypoxic-ischemic injury: sonography and dynamic color Doppler sonography perfusion of the brain and abdomen with pathologic correlation. American Journal of Roentgenology. 199 (6), 743-752 (2012).
- Shen, Q., Stuart, J., Venkatesh, B., Wallace, J., Lipman, J. Inter observer variability of the transcranial Doppler ultrasound technique: impact of lack of practice on the accuracy of measurement. Journal of Clinical Monitoring and Computing. 15 (3-4), 179-184 (1999).
Cite This Article
Neulen, A., Molitor, M., Kosterhon, M., Pantel, T., Karbach, S. H., Wenzel, P., Gaul, T., Ringel, F., Thal, S. C. Analysis of Cerebral Vasospasm in a Murine Model of Subarachnoid Hemorrhage with High Frequency Transcranial Duplex Ultrasound. J. Vis. Exp. (172), e62186, doi:10.3791/62186 (2021).