使用微透析系统实时动态收集意识大鼠的海马细胞外液
1Innovative Institute of Chinese Medicine and Pharmacy,Chengdu University of Traditional Chinese Medicine, 2Research Institute of Integrated TCM & Western Medicine,Chengdu University of Traditional Chinese Medicine, 3School of Pharmacy,Chengdu University of Traditional Chinese Medicine, 4School of Ethnic Medicine,Chengdu University of Traditional Chinese Medicine
Summary
这里的协议提供了使用微透析系统从清醒大鼠海马体中细胞外液的详细实时动态采样。
Abstract
多种中枢神经系统（CNS）疾病与海马细胞外液（HECF）组成的变化有关。然而，从有意识的大鼠中实时获得HECF的困难长期以来限制了CNS疾病进展的评估和民族药物治疗的有效性。令人鼓舞的是，脑微透析技术可用于连续采样，具有动态观察、定量分析和小采样量的优点。这允许监测活体动物大脑中传统草药化合物及其代谢物的细胞外液含量的变化。因此，本研究的目的是用三维脑立体定位装置将脑脊液微透析探针准确地植入Sprague Dawley（SD）大鼠的海马区域，切断大于20kDa的分子量。然后使用微透析采样控制系统从意识大鼠中获得高质量的HECF，采样速率可调为2.87 nL / min – 2.98 mL / min。综上所述，我们的方案提供了一种高效、快速、动态的方法，借助微透析技术在清醒大鼠中获取HECF，为进一步探索中枢神经系统相关疾病的发病机制和评估药物疗效提供了无限的可能性。
Introduction
高发病率的中枢神经系统（CNS）疾病，如神经退行性疾病、创伤性脑损伤、高原缺氧引起的脑损伤和缺血性脑卒中，是全球死亡率上升的主要原因1，2，3。实时监测特定大脑区域的细胞因子和蛋白质变化有助于CNS疾病的诊断准确性和用药后脑药代动力学研究。传统的科学研究使用脑组织匀浆或手动收集动物间质脑液来检测特定物质和进行药代动力学研究。但是，这有一些缺点，例如样本量有限，无法动态观察指标的变化以及采样质量不均匀4，5，6。脑脊液是一种间质液，可保护大脑和脊髓免受机械损伤。由于血脑屏障（BBB）的存在，其成分与血清的组成不同7。直接分析脑脊液样本更有利于揭示中枢神经系统病变机制和药物发现。不可避免地，通过注射器直接从大池和脑室手动获得的脑脊液样本具有血液污染，样本采集机会随机，数量不确定以及几乎没有多次检索可能性的缺点8，9。更值得注意的是，传统的间质脑液采样方法无法从受损脑区域获取样本，这阻碍了对特定脑区中枢神经系统疾病的发病机制的探索和靶向民族医学治疗的疗效评估9，10。
脑微透析是一种对清醒动物的间质脑液进行采样的技术11。微透析系统借助植入大脑的探针模仿血管通透性。微透析探针配备有半透膜，并植入特定的大脑区域。用等渗人工脑脊液（ACSF）灌注后，透析的间质脑液可以有利地收集，具有小样本量，连续采样和动态观察的优点12，13。就位置而言，脑微透析探针可以选择性地植入感兴趣的脑结构或颅骨池中14。观察到海马细胞外液（HECF）中内源性物质的异常水平提示中枢神经系统疾病的发生或疾病的发病机制。多项研究表明，中枢神经系统疾病的生物标志物，如精神分裂症中的D-氨基酸，阿尔茨海默病中的β-淀粉样蛋白和tau蛋白，创伤性脑损伤中的神经丝轻链，以及缺氧缺血性脑病中的泛素羧基末端水解酶L1s，可以在脑脊液中分析15，16，17.基于脑微透析采样技术的化学分析方法可用于监测外源性化合物（例如民族医学的活性成分）的动态变化，这些化合物在特定大脑区域扩散和分布14。
本文介绍了清醒大鼠动态HECF采集的具体过程，并测量其渗透压以确保样品质量。
Protocol
实验方案按照成都中医药大学实验动物使用和机构动物护理使用委员会的要求进行（备案号：2021-11）。雄性Sprague Dawley（SD）大鼠（280±20g，6-8周龄）用于本研究。 1.脑微透析探针植入手术 分别使用3%和1.5%异氟醚诱导和维持大鼠麻醉，使用动物麻醉系统在空气 – 氧气混合物中以0.6L / min的速度进行。确保大鼠被深度麻醉，没有疼痛反射和角膜反射。在眼睛上…
Representative Results
按照上述实验方案和 表1中设置的采样参数，以设定的采样速率获得水状，无色和透明的大鼠HECF（图1K）。所得大鼠HECF的渗透压为290-310 mOsm/L，可间接保证样品18，19的质量。 图1<…
Discussion
中枢神经系统疾病的发病机制尚未完全了解，这阻碍了新疗法和药物的开发。研究表明，大多数中枢神经系统疾病与海马病变密切相关20，21，22。所提出的脑微透析技术可以针对大脑的特定区域，尤其是海马体，这使得它从传统的收集HECF的方法中脱颖而出。通过植入手术将探针放置在大鼠大脑的CA1区域，以通过人造膜的被动?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国家自然科学基金（82104533）、四川省科技厅（2021YJ0175）和中国博士后科学基金（2020M683273）的支持。作者要感谢Tri-Angels D&H Trading Pte的高级设备工程师Yuncheng Hong先生。Ltd.（新加坡）为微透析技术提供技术服务。
Materials
| Air-drying oven | Suzhou Great Electronic Equipment Co., Ltd | GHG-9240A | |
| Animal anesthesia system | Rayward Life Technology Co., Ltd | R500IE | |
| Animal temperature maintainer | Rayward Life Technology Co., Ltd | 69020 | |
| Artificial cerebrospinal fluid | Beijing leagene biotech. Co., Ltd | CZ0522 | |
| Brain microdialysis probe | CMA Microdialysis AB | T56518 | |
| Catheter | CMA Microdialysis AB | T56518 | |
| Covance infusion harness | Instech Laboratories, Inc. | CIH95 | |
| Denture base resins | Shanghai Eryi Zhang Jiang Biomaterials Co., Ltd | 190732 | |
| Electric cranial drill | Rayward Life Technology Co., Ltd | 78001 | |
| Electric shaver | Rayward Life Technology Co., Ltd | CP-5200 | |
| Free movement tank for animals | CMA Microdialysis AB | CMA120 | |
| Heparin sodium injection | Chengdu Haitong Pharmaceutical Co., Ltd | H51021208 | |
| Iodophor | Sichuan Lekang Pharmaceutical Accessories Co., Ltd | 202201 | |
| Isofluran | Rayward Life Technology Co., Ltd | R510-22 | |
| Microdialysis catheter stylet | CMA Microdialysis AB | 8011205 | |
| Microdialysis collection tube | CMA Microdialysis AB | 7431100 | |
| Microdialysis collector | CMA Microdialysis AB | CMA4004 | |
| Microdialysis fep tubing | CMA Microdialysis AB | 3409501 | |
| Microdialysis in vitro stand | CMA Microdialysis AB | CMA130 | |
| Microdialysis microinjection pump | CMA Microdialysis AB | 788130 | |
| Microdialysis syringe (1.0 mL) | CMA Microdialysis AB | 8309020 | |
| Microdialysis tubing adapter | CMA Microdialysis AB | 3409500 | |
| Non-absorbable surgical sutures | Shanghai Tianqing Biological Materials Co., Ltd | S19004 | |
| Ophthalmic forceps | Rayward Life Technology Co., Ltd | F12016-15 | |
| Osmometer | Löser | OM 807 | |
| Sodium hyaluronate eye drops | URSAPHARM Arzneimittel GmbH | H20150150 | |
| Stereotaxie apparatus | Rayward Life Technology Co., Ltd | 68025 | |
| Surgical scissors | Rayward Life Technology Co., Ltd | S14014-15 | |
| Surgical scissors | Shanghai Bingyu Fluid technology Co., Ltd | BY-103 | |
| Syringe needle | CMA Microdialysis AB | T56518 | |
| Trypsin solution | Boster Biological Technology, Ltd. |
PYG0107 | |
| Ultrasonic cleaner | Guangdong Goote Ultrasonic Co., Ltd | KMH1-240W8101 |
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Wang, X., Xie, N., Zhang, Y., Meng, X., Hou, Y., Zhang, S. Real-Time Dynamic Collection of Hippocampal Extracellular Fluid from Conscious Rats Using a Microdialysis System. J. Vis. Exp. (188), e64530, doi:10.3791/64530 (2022).