在脑电图记录期间从大鼠的侧尾静脉采集脑脊液和血液
Summary
该协议显示癫痫大鼠的重复脑脊液和血液采集与连续视频脑电图（EEG）监测同时进行。这些有助于探索各种体液分子变化与癫痫发作活动之间的可能联系。
Abstract
由于体液的成分反映了许多生理和病理动力学，因此在许多实验环境中通常获得生物液体样品来测量感兴趣的分子，例如激素、生长因子、蛋白质或小的非编码 RNA。一个具体的例子是在癫痫生物标志物研究中对生物液体进行采样。在这些研究中，希望通过同时提取脑脊液和血浆并考虑从癫痫发作到癫痫发作的采样时间距离来比较脑脊液 （CSF） 和血浆中的分子水平。脑脊液和血浆取样相结合，加上癫痫动物的视频脑电图监测，是验证推定诊断和预后生物标志物的一种有前途的方法。在这里，描述了从大池中撤出脑脊液和从连续视频脑电图监测的癫痫大鼠的侧尾静脉取血的联合程序。与其他常用技术相比，该程序具有显着优势。它允许以最小的疼痛或侵入性快速取样，并减少麻醉时间。此外，它可用于在系留和遥测脑电图记录的大鼠中获取脑脊液和血浆样本，并且可以在多天的实验中重复使用。通过缩短异氟烷麻醉时间，最大限度地减少取样引起的压力，预计测量将更准确地反映生物流体中所研究分子的真实水平。根据适当分析测定的可用性，该技术可用于测量多个不同分子的水平，同时进行脑电图记录。
Introduction
在临床前和临床研究中，脑脊液 （CSF） 和血液采样对于识别和验证癫痫的生物标志物都很重要 1,2。如今，癫痫的诊断和大多数关于癫痫生物标志物的研究都集中在脑电图和神经影像学上 3,4,5。然而，这些方法存在一些局限性。除了常规的头皮测量外，在许多情况下，脑电图还需要深度电极等侵入性技术6.脑成像方法的时间和空间分辨率较差，并且相对昂贵且耗时 7,8。出于这个原因，识别非侵入性、低成本和基于生物流体的生物标志物将提供一种非常有吸引力的替代方案。此外，这些生物流体生物标志物可以与现有的诊断方法相结合，以提高其预测性。
被诊断为癫痫的患者通常接受脑电图9、10 和血液采样11、12、13、14 检查，许多患者还接受脑脊液停药检查，以排除危及生命的病因（即急性感染、自身免疫性脑炎）15。这些血液和脑脊液样本可用于临床研究，旨在识别癫痫的生物标志物。例如，Hogg及其同事发现，在人类癫痫中，三个血浆tRNA片段的增加先于癫痫发作14。同样，人脑脊液和血清中的白细胞介素-1β （IL-1β） 水平，表示为脑脊液中 IL-1β 水平与血清的比率，可以预测创伤性脑损伤后创伤后癫痫的发展16.这些研究强调了生物流体采样对癫痫生物标志物研究的重要性，但它们面临着临床试验固有的多种局限性，例如，血液中抗癫痫药物 （AED） 的共同创始因素、经常缺乏病因信息、控制不足、患者数量适中等17,18。
临床前研究为研究生物流体中的分子作为癫痫的潜在生物标志物提供了其他机会。事实上，在进行脑电图记录时，可以从动物身上提取血浆和/或脑脊液。此外，可以在多天的实验中重复进行采样，并且可以使用一些年龄、性别和癫痫侮辱匹配的对照来提高研究的稳健性。在这里，详细描述了一种从脑电图监测的大鼠尾静脉平行抽取血浆从大池获得脑脊液的灵活技术。与其他方法相比，所提出的技术有几个优点。通过使用蝶形针方法，可以在不影响脑电图电极或类似头部植入物功能的情况下多次收集脑脊液。这代表了鞘内导管拔出手术的改进，鞘内导管拔出手术与相对较高的感染风险相关。此外，据报道，用于采血的自由落体方法优于其他尾静脉抽血方法，因为血液不通过管道且不施加真空压力，溶血风险大大降低。如果在严格的无菌条件下进行，动物感染的风险特别低。此外，通过从动物尾巴的末端开始抽血，可以重复采样几次。这些技术很容易掌握，可以应用于许多中枢神经系统疾病的临床前研究。
Protocol
根据 1986 年 11 月 24 日欧洲共同体理事会关于保护用于实验和其他科学目的的动物的指令 （86/609/EEC） 中概述的指南，所有实验程序均已获得费拉拉大学机构动物护理和使用委员会和意大利卫生部的批准（授权：DM 603/2022-PR）。该方案专门针对在大鼠脑脊液和癫痫动物脑电图控制下获得的血浆中小的非编码核糖核酸（sncRNA）的进一步定量聚合酶链反应（qPCR）分析进行了调整。根据其选择，请参阅?…
Representative Results
在 9 只对照组和 18 只慢性癫痫大鼠中进行的不同 CSF 和抽血手术的结果，均在 SE 后 1 个月植入电极，以成功率报告。植入后，对所有大鼠进行视频脑电图监测1个月，在此期间，在实验的最后两周（即在SE后第52、55、58、61和64天）每3天抽取5次脑脊液加血。来自不同动物的多次拔出的数据用于比较双头植入物捐赠大鼠（插管用于脑脊液退出）的脑脊液采集成功率与仅栓系或遥测电极植入动物的脑脊…
Discussion
目前的工作说明了一种易于掌握的大鼠脑脊液和采血技术，该技术不仅可能对癫痫模型的研究有用，而且对其他神经系统疾病或疾病（如阿尔茨海默病、帕金森病或多发性硬化症）的研究也很有用。在癫痫研究中，当追求不同可溶性分子水平与癫痫发作活动之间的相关性时，两种采样程序与视频脑电图相结合都是理想的。出于这个特定原因，采用了连续的视频脑电图记录：i） 为了正确诊断癫痫?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究得到了欧盟地平线 2020 工作计划（称为 H2020-FETOPEN-2018-2020）的赠款支持，赠款协议 964712 （PRIME; 给 M. Simonato）。
Materials
| Blood collection set BD Vacutainer Safety-Lok | BD Italy SpA, Milan, Italy | 367246 | Material |
| Blood Collection tubes (Microtainer K2E) | BD Italy SpA, Milan, Italy | 365975 | Material |
| Butterfly Winged Infusion Set 23G x 3/4'' 0.6 x 19 mm | Nipro, Osaka, Japan | PSY-23-ET-ICU | Material |
| Centrifuge refrigerated ALC PK 130R | DJB Labcare Ltd, Buckinghamshire, England | 112000033 | Material |
| Cotton suture 3-0 | Ethicon, Johnson & Johnson surgical technologies, Raritan, New Jersey, USA | 7343H | Material |
| Diazepam 5 mg/2ml, Solupam | Dechra Veterinary Products, Torino, Italy | 105183014 (AIC) | Solution |
| Digital video 8-channel media recorder system of telemetry EEG set up | Data Sciences International (DSI), St Paul, MN, USA | PNM-VIDEO-008 | Equipment |
| Digital video surveillance system of tethered EEG set up | EZVIZ Network, Hangzhou, Cina | EZVIZ (V5.3.2) | Equipment |
| Disinfectant based on stabilized peroxides and quaternary ammonium activity | Laboratoire Garcin-Bactinyl, France | LB 920111 | Solution |
| Dummy guide cannula 8 mm | Agn Tho's, Lindigö, Sweden | CXD-8 | Material |
| Electrode 3-channel two-twisted | Invivo1, Plastic One, Roanoke, Virginia, USA | MS333/3-B/SPC | Material |
| Electrode holder for stereotxic surgery | Agn Tho's, Lindigö, Sweden | 1776-P1 | Equipment |
| Eppendorf BioSpectrometer basic | Eppendorf AG, Hamburg, Germany | 6137 | Equipment |
Eppendorf PCR Tubes 0.2 mL |
Eppendorf Srl, Milan, Italy | 30124332 | Material |
| Eppendorf μCuvette G1.0 | Eppendorf AG, Hamburg, Germany | 6138 | Equipment |
| Feeding needle flexible 17G for rat | Agn Tho's, Lindigö Sweden | 7206 | Material |
| Grass Technology apparatus | Grass Technologies, Natus Neurology Incorporated, Pleasanton, California, USA | M665G08 | Equipment (AS40 amplifier, head box, interconnecting cables, telefactor model RPSA S40) |
| Isoflurane 100%, IsoFlo | Zoetis, Rome, Italy | 103287025 (AIC) | Solution |
| Ketamine (Imalgene) | Merial, Toulouse, France | 221300288 (AIC) | Solution |
| Lithium chloride | Sigma-Aldrich, Milan, Italy | L9650 | Material |
| Microinjection cannula 31G 9 mm | Agn Tho's, Lindigö Sweden | CXMI-9 | Material |
| MP150 modular data acquisition and analysis system | Biopac, Goleta, California, USA | MP150WSW | Equipment |
| Ophthalmic vet ointment, Hylo night | Ursapharm, Milan, Italy | 941791927 (AIC) | Material |
| Pilocarpine hydrochloride | Sigma-Aldrich, Milan, Italy | P6503 | Material |
| PTFE Tube with joint | Agn Tho's, Lindigö, Sweden | JT-10 | Material |
| Saline | 0.9% NaCl, pH adjusted to 7.0 | Solution | |
| Scopolamine hydrobromide trihydrate | Sigma-Aldrich, Milan, Italy | S2250 | Material |
| Scopolamine methyl nitrate | Sigma-Aldrich, Milan, Italy | S1876 | Material |
| Silver sulfadiazine 1% cream | Sofar, Trezzano Rosa, Milan, Italy | 025561010 (AIC) | Material |
| Simplex rapid dental methacrylic cement | Kemdent, Associated Dental Products Ltd, Swindon, United Kingdom | ACR811 | Material |
| Stereotaxic apparatus | David Kopf Instruments, Los Angeles, CA, USA | Model 963 | Equipment |
| Sucrose solution | 10% sucrose in distilled water | Home-made | Solution |
| Syringe 1 mL | Biosigma, Cona, Venezia, Italy | 20,71,26,03,00,350 | Material |
| Telemeters | Data Sciences International (DSI), St Paul, MN, USA | CTA-F40 | Material |
| Telemetry EEG traces analyzer | Data Sciences International (DSI), St Paul, MN, USA | NeuroScore v3-0 | Equipment |
| Telemetry system | Data Sciences International (DSI), St Paul, MN, USA | Hardware plus software Ponemah core 6.51 | Equipment |
| Xylazine hydrochloride | Sigma-Aldrich, Milan, Italy | X1251 | Material |
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Cite This Article
Soukupová, M., Guarino, A., Asth, L., Marino, P., Barbieri, M., Simonato, M., Zucchini, S. Sampling Cerebrospinal Fluid and Blood from Lateral Tail Vein in Rats During EEG Recordings. J. Vis. Exp. (199), e65636, doi:10.3791/65636 (2023).