纤维蛋白富集和 tPA 敏感的光血栓形成卒中模型
概要
传统的光血栓中风 （PTS） 模型主要诱导对组织纤溶酶原激活剂 （tPA） 溶解治疗具有高耐药性的致密血小板聚集体。本文通过共注射凝血酶和光敏染料进行光活化，引入了改良的小鼠PTS模型。凝血酶增强的 PTS 模型产生混合血小板：纤维蛋白凝块，并且对 tPA 溶栓高度敏感。
Abstract
理想的血栓栓塞性卒中模型需要某些特性，包括相对简单的外科手术、死亡率低、梗死大小和位置一致、血小板：纤维蛋白混合血凝块的沉淀与患者相似，以及对纤溶治疗具有足够的敏感性。基于孟加拉玫瑰 （RB） 染料的光血栓中风模型满足前两个要求，但对 tPA 介导的裂解处理高度难治，这可能是由于其富含血小板但纤维蛋白贫乏的凝块组成。我们认为，RB染料（50mg / kg）和亚血栓形成剂量的凝血酶（80 U / kg）的组合用于针对大脑中动脉（MCA）近端分支的光活化，可能会产生富含纤维蛋白和tPA敏感的凝块。事实上，凝血酶和RB（T+RB）联合光血栓形成模型触发了混合血小板：纤维蛋白血凝块，如免疫染色和免疫印迹所示，并保持一致的梗死大小和位置以及低死亡率。此外，在光激活后 2 小时内静脉注射 tPA（阿替普酶，10 mg/kg）可显着减小 T+RB 光血栓形成的梗死面积。因此，凝血酶增强的光血栓中风模型可能是测试新型溶栓疗法的有用实验模型。
Introduction
血管内血栓切除术和 tPA 介导的溶栓是美国食品和药物管理局 （FDA） 批准的仅有的两种急性缺血性卒中疗法，美国每年有 ~700,000 名患者患病1。由于血栓切除术的应用仅限于大血管闭塞 （LVO），而 tPA 溶栓术可以缓解小血管闭塞，因此两者都是急性缺血性卒中2 的有价值的治疗方法。此外，两种疗法的联合使用（例如，在卒中发作后 4.5 小时内开始 tPA 溶栓，然后进行血栓切除术）可改善再灌注和功能结局3。因此，即使在血栓切除术时代，优化溶栓仍然是卒中研究的重要目标。
血栓栓塞模型是临床前卒中研究的重要工具，旨在改善溶栓治疗。这是因为机械血管闭塞模型（例如，腔内缝合 MCA 闭塞）不会产生血凝块，并且其在去除机械闭塞后脑血流的快速恢复被过度理想化 4,5。迄今为止，主要的血栓栓塞模型包括光血栓形成 6,7,8、局部氯化铁 （FeCl 3） 应用9、凝血酶显微注射到 MCA 分支 10,11、体外（微）栓子注射到 MCA 或颈总动脉 （CCA） 12、13、14 和短暂性缺氧缺血 （tHI）15,16，17,18.这些卒中模型在随后血栓的组织学组成和对tPA介导的裂解疗法的敏感性方面有所不同（表1）。它们在开颅手术要求（原位凝血酶注射和局部应用 FeCl3 需要）、梗死大小和位置的一致性（例如，CCA 输注微栓子产生非常可变的结果）以及对心血管系统的总体影响（例如，tHI 增加心率和心输出量以补偿缺氧诱导的外周血管舒张）方面也有所不同。
基于 RB 染料的光血栓卒中 （PTS） 模型具有许多吸引人的特征，包括简单的无开颅手术、低死亡率（通常< 5%）以及可预测的梗死大小和位置（在 MCA 供应区域），但它有两个主要局限性。8 首先需要注意的是，对 tPA 介导的溶栓治疗反应弱至零，这也是 FeCl3 模型7、19、20 的缺点。PTS 和 FeCl3 卒中模型的第二个警告是，随后的血栓由密集的血小板聚集体和少量纤维蛋白组成，这不仅导致其对 tPA 溶解治疗的弹性，而且还偏离了急性缺血性卒中患者中血小板：纤维蛋白血栓混合的模式21,22。相比之下，原位凝血酶显微注射模型主要包括聚合纤维蛋白和不确定含量的血小板10。
鉴于上述推理，我们假设 RB 和亚血栓形成剂量的凝血酶的混合物通过变薄的颅骨进行 MCA 靶向光活化可能会增加所得血栓中的纤维蛋白成分并提高对 tPA 介导的裂解治疗的敏感性。我们已经证实了这一假设，23 在此我们描述了改良 （T+RB） 光血栓中风模型的详细程序。
Protocol
Representative Results
Discussion
1985 年引入的传统 RB 光血栓性卒中是一种有吸引力的局灶性脑缺血模型，适用于简单的外科手术、低死亡率和高可重复性脑梗死。5 在该模型中，光动力染料 RB 在光激发下迅速激活血小板，导致致密的聚集体阻塞血管 5,8,23。然而，RB诱导的血凝块中的少量纤维蛋白（图2）偏离了缺血性?…
開示
The authors have nothing to disclose.
Acknowledgements
这项工作得到了美国国立卫生研究院（NIH）的资助（NS108763、NS100419、NS095064和HD080429给CYK和NS106592给YYS）。
Materials
| 2,3,5-triphenyltetrazolium chloride (TTC) | Sigma | T8877 | infarct |
| 4-0 Nylon monofilament suture | LOOK | 766B | surgical supplies |
| 5-0 silk suture | Harvard Apparatus | 624143 | surgical supplies |
| 543nm laser beam | Melles Griot | 25-LGP-193-249 | photothrombosis |
| adult male mice | Charles River | C57BL/6 | 10~14 weeks old (22~30 g) |
| Anesthesia bar for mouse adaptor | machine shop, UVA | surgical setup | |
| Avertin (2, 2, 2-Tribromoethanol) | Sigma | T48402 | euthanasia |
| Dental drill | Dentamerica | Rotex 782 | surgical setup |
| Digital microscope | Dino-Lite | AM2111 | brain imaging |
| Dissecting microscope | Olympus | SZ40 | surgical setup |
| Fine curved forceps (serrated) | FST | 11370-31 | surgical instrument |
| Fine curved forceps (smooth) | FST | 11373-12 | surgical instrument |
| goat anti-rabbit Alexa Fluro 488 | Invitrogen | A11008 | Immunohistochemistry |
| Halsted-Mosquito hemostats | FST | 13008-12 | surgical instrument |
| Heat pump with warming pad | Gaymar | TP700 | surgical setup |
| infusion pump | KD Scientific | 200 | thrombolytic treatment |
| Insulin syringe with 31G needle | BD | 328291 | photothrombosis |
| Ketamine | CCM, UVA | anesthesia | |
| Laser protective google 532nm | Thorlabs | LG3 | photothrombosis |
| Meloxicam SR | CCM, UVA | NSAID analgesia | |
| micro needle holders | FST | 12060-01 | surgical instrument |
| micro scissors | FST | 15000-03 | surgical instrument |
| MoorFLPI-2 blood flow imager | Moor | 780-nm laser source | Laser Speckle Contrast Imaging |
| Mouse adaptor | RWD | 68014 | surgical setup |
| Puralube Vet ointment | Fisher | NC0138063 | eye dryness prevention |
| Retractor tips | Kent Scientific | Surgi-5014-2 | surgical setup |
| Rose Bengal | Sigma | 198250 | photothrombosis |
| Thrombin | Sigma | T7513 | photothrombosis |
| Tissue glue | Abbott Laboratories | NC9855218 | surgical supplies |
| tPA | Genetech | Cathflo activase 2mg | thrombolytic treatment |
| Vibratome | Stoelting | 51425 | TTC infacrt |
| Xylazine | CCM, UVA | anesthesia |
参考文献
- Lyden, P. D. . Thrombolytic Therapy for Acute Stroke. 3/e. , (2015).
- Linfante, I., Cipolla, M. J. Improving reperfusion therapies in the era of mechanical thrombectomy. Translational Stroke Research. 7 (4), 294-302 (2016).
- Campbell, B. C., et al. Endovascular Therapy for Ischemic stroke with perfusion-imaging selection. The New England Journal of Medicine. 372 (11), 1009-1018 (2015).
- Hossmann, K. A. The two pathophysiologies of focal brain ischemia: implications for translational stroke research. Journal of Cerebral Blood Flow and Metabolism. 32 (7), 1310-1316 (2012).
- Longa, E. Z., Weinstein, P. R., Carlson, S., Cummins, R. Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke. 20 (1), 84-91 (1989).
- Watson, B. D., Dietrich, W. D., Busto, R., Wachtel, M. S., Ginsberg, M. D. Induction of reproducible brain infarction by photochemically initiated thrombosis. Annals of Neurology. 17 (5), 497-504 (1985).
- Watson, B. D., Prado, R., Veloso, A., Brunschwig, J. P., Dietrich, W. D. Cerebral blood flow restoration and reperfusion injury after ultraviolet laser-facilitated middle cerebral artery recanalization in rat thrombotic stroke. Stroke. 33 (2), 428-434 (2002).
- Uzdensky, A. B. Photothrombotic stroke as a model of ischemic stroke. Translational Stroke Research. 9 (5), 437-451 (2018).
- Karatas, H., et al. Thrombotic distal middle cerebral artery occlusion produced by topical FeCl(3) application: a novel model suitable for intravital microscopy and thrombolysis studies. Journal of Cerebral Blood Flow and Metabolism. 31 (3), 1452-1460 (2011).
- Orset, C., et al. Mouse model of in situ thromboembolic stroke and reperfusion. Stroke. 38 (10), 2771-2778 (2007).
- Orset, C., et al. Efficacy of Alteplase in a mouse model of acute ischemic stroke: A retrospective pooled analysis. Stroke. 47 (5), 1312-1318 (2016).
- Kudo, M., Aoyama, A., Ichimori, S., Fukunaga, N. An animal model of cerebral infarction. Homologous blood clot emboli in rats. Stroke. 13 (4), 505-508 (1982).
- Busch, E., Kruger, K., Hossmann, K. A. Improved model of thromboembolic stroke and rt-PA induced reperfusion in the rat. Brain Research. 778 (1), 16-24 (1997).
- Lapchak, P. A., Araujo, D. M., Zivin, J. A. Comparison of Tenecteplase with Alteplase on clinical rating scores following small clot embolic strokes in rabbits. Experimental Neurology. 185 (1), 154-159 (2004).
- Sun, Y. Y., et al. Synergy of combined tPA-Edaravone therapy in experimental thrombotic stroke. PLoS One. 9 (6), 98807 (2014).
- Sun, Y. Y., et al. Prophylactic Edaravone prevents transient hypoxic-ischemic brain injury: Implications for perioperative neuroprotection. Stroke. 46 (7), 1947-1955 (2015).
- Sun, Y. Y., et al. Sickle mice are sensitive to hypoxia/ischemia-induced stroke but respond to tissue-type plasminogen activator treatment. Stroke. 48 (12), 3347-3355 (2017).
- Sun, Y. Y., Kuan, C. Y. A thrombotic stroke model based on transient cerebral hypoxia-ischemia. Journal of Visualized Experiments. (102), e52978 (2015).
- Pena-Martinez, C., et al. Pharmacological modulation of neutrophil extracellular traps reverses thrombotic stroke tPA (tissue-type plasminogen activator) resistance. Stroke. 50 (11), 3228-3237 (2019).
- Denorme, F., et al. ADAMTS13-mediated thrombolysis of t-PA-resistant occlusions in ischemic stroke in mice. Blood. 127 (19), 2337-2345 (2016).
- Marder, V. J., et al. Analysis of thrombi retrieved from cerebral arteries of patients with acute ischemic stroke. Stroke. 37 (8), 2086-2093 (2006).
- Bacigaluppi, M., Semerano, A., Gullotta, G. S., Strambo, D. Insights from thrombi retrieved in stroke due to large vessel occlusion. Journal of Cerebral Blood Flow and Metabolism. 39 (8), 1433-1451 (2019).
- Sun, Y. Y., et al. A murine photothrombotic stroke model with an increased fibrin content and improved responses to tPA-lytic treatment. Blood Advances. 4 (7), 1222-1231 (2020).
- Su, E. J., et al. Activation of PDGF-CC by tissue plasminogen activator impairs blood-brain barrier integrity during ischemic stroke. Nature Medicine. 14 (7), 731-737 (2008).
- Gupta, A. K., et al. Protective effects of gelsolin in acute pulmonary thromboembolism and thrombosis in the carotid artery of mice. PLoS One. 14 (4), 0215717 (2019).
- Carroll, B. J., Piazza, G. Hypercoagulable states in arterial and venous thrombosis: When, how, and who to test. Vascular Medicine. 23 (4), 388-399 (2018).
- Coutts, S. B., Berge, E., Campbell, B. C., Muir, K. W., Parsons, M. W. Tenecteplase for the treatment of acute ischemic stroke: A review of completed and ongoing randomized controlled trials. International Journal of Stroke. 13 (9), 885-892 (2018).
- McFadyen, J. D., Schaff, M., Peter, K. Current and future antiplatelet therapies: emphasis on preserving haemostasis. Nature Reviews Cardiology. 15 (3), 181-191 (2018).
- Bang, O. Y., Goyal, M., Liebeskind, D. S. Collateral crculation in ischemic stroke: Assessment tools and therapeutic strategies. Stroke. 46 (11), 3302-3309 (2015).
- Faber, J. E., Chilian, W. M., Deindl, E., van Royen, N., Simons, M. A brief etymology of the collateral circulation. Arteriosclerosis, Thrombsis, Vascular Biology. 34 (9), 1854-1859 (2014).
