Summary

使用微透析系统实时动态收集意识大鼠的海马细胞外液

Published: October 21, 2022
doi:

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123。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4,56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89。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910

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1213。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151617.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202122。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

Referências

  1. Erkkinen, M. G., Kim, M. O., Geschwind, M. D. Clinical neurology and epidemiology of the major neurodegenerative diseases. Cold Spring Harbor Perspectives in Biology. 10 (4), 033118 (2018).
  2. Salehi, A., Zhang, J. H., Obenaus, A. Response of the cerebral vasculature following traumatic brain injury. Journal of Cerebral Blood Flow and Metabolism. 37 (7), 2320-2339 (2017).
  3. Kurtzman, R. A. e. m. 3., Caruso, J. L. High-altitude illness death investigation. Academic Forensic Pathology. 8 (1), 83-97 (2018).
  4. Matsumoto, T., et al. Pharmacokinetic study of Ninjin’yoeito: Absorption and brain distribution of Ninjin’yoeito ingredients in mice. Journal of Ethnopharmacology. 279, 114332 (2021).
  5. Mahat, M. Y., et al. An improved method of transcutaneous cisterna magna puncture for cerebrospinal fluid sampling in rats. Journal of Neuroscience Methods. 211 (2), 272-279 (2012).
  6. Ceaglio, N., et al. High performance collection of cerebrospinal fluid in rats: evaluation of erythropoietin penetration after osmotic opening of the blood-brain barrier. Journal of Neuroscience Methods. 219 (1), 70-75 (2013).
  7. Bothwell, S. W., Janigro, D., Patabendige, A. Cerebrospinal fluid dynamics and intracranial pressure elevation in neurological diseases. Fluids and Barriers of the CNS. 16 (1), 9 (2019).
  8. Barthel, L., et al. A step-by-step guide for microsurgical collection of uncontaminated cerebrospinal fluid from rat cisterna magna. Journal of Neuroscience Methods. 352, 109085 (2021).
  9. Zhao, Y., Yang, Y., Wang, D. X., Wang, J., Gao, W. Y. Cerebrospinal fluid amino acid metabolite signatures of diabetic cognitive dysfunction based on targeted mass spectrometry. Journal of Alzheimer’s Disease. 86 (4), 1655-1665 (2022).
  10. Lim, N. K., et al. An improved method for collection of cerebrospinal fluid from anesthetized mice. Journal of Visualized Experiments. (133), e56774 (2018).
  11. Hendrickx, S., et al. A sensitive capillary LC-UV method for the simultaneous analysis of olanzapine, chlorpromazine and their FMO-mediated N-oxidation products in brain microdialysates. Talanta. 162, 268-277 (2017).
  12. Chefer, V. I., Thompson, A. C., Zapata, A., Shippenberg, T. S. Overview of brain microdialysis. Current Protocols in Neuroscience. , (2009).
  13. Hammarlund-Udenaes, M. Microdialysis as an important technique in systems pharmacology-a historical and methodological review. The AAPS Journal. 19 (5), 1294-1303 (2017).
  14. Anderzhanova, E., Wotjak, C. T. Brain microdialysis and its applications in experimental neurochemistry. Cell and Tissue Research. 354 (1), 27-39 (2013).
  15. Mohammadi, A., Rashidi, E., Amooeian, V. G. Brain, blood, cerebrospinal fluid, and serum biomarkers in schizophrenia. Psychiatry Research. 265, 25-38 (2018).
  16. Lashley, T., et al. Molecular biomarkers of Alzheimer’s disease: progress and prospects. Disease Models & Mechanisms. 11 (5), 031781 (2018).
  17. Kawata, K., Tierney, R., Langford, D. Blood and cerebrospinal fluid biomarkers. Handbook of Clinical Neurology. 158, 217-233 (2018).
  18. Zhao, Q. P., et al. Protective effects of dehydrocostuslactone on rat hippocampal slice injury induced by oxygen-glucose deprivation/reoxygenation. International Journal of Molecular Medicine. 42 (2), 1190-1198 (2018).
  19. Wang, X. B. . Protective effects of dehydrocostuslactone on oxygen-glucose deprivation injury in rat hippocampal slices. , (2017).
  20. Coimbra-Costa, D., Alva, N., Duran, M., Carbonell, T., Rama, R. Oxidative stress and apoptosis after acute respiratory hypoxia and reoxygenation in rat brain. Redox Biology. 12, 216-225 (2017).
  21. Liu, H. Y., Chou, K. H., Chen, W. T. Migraine and the Hippocampus. Current Pain and Headache Reports. 22 (2), 13 (2018).
  22. Toda, T., Parylak, S. L., Linker, S. B., Gage, F. H. The role of adult hippocampal neurogenesis in brain health and disease. Molecular Psychiatry. 24 (1), 67-87 (2019).
  23. Wang, P., Lo Cascio, F., Gao, J., Kayed, R., Huang, X. F., F, X. Binding and neurotoxicity mitigation of toxic tau oligomers by synthetic heparin like oligosaccharides. Chemical Communications. 54 (72), 10120-10123 (2018).
  24. Han, J. Y., Li, Q., Ma, Z. Z., Fan, J. Y. Effects and mechanisms of compound Chinese medicine and major ingredients on microcirculatory dysfunction and organ injury induced by ischemia/reperfusion. Pharmacology & Therapeutics. 177, 146-173 (2017).
  25. Peng, T. M., et al. Anti-inflammatory effects of traditional Chinese medicines on preclinical in vivo models of brain ischemia-reperfusion-injury: Prospects for neuroprotective drug discovery and therapy. Frontiers in Pharmacology. 10, 204 (2019).
  26. König, M., Thinnes, A., Klein, J. Microdialysis and its use in behavioural studies: Focus on acetylcholine. Journal of Neuroscience Methods. 300, 206-215 (2018).
  27. Liu, M. Z., Wang, P., Yu, X. M., Dong, G. C., Yue, J. Intracerebral microdialysis coupled to LC-MS/MS for the determination tramadol and its major pharmacologically active metabolite O-desmethyltramadol in rat brain microdialysates. Drug Testing and Analysis. 9 (8), 1243-1250 (2017).
  28. de Lima Oliveira, M., et al. Cerebral microdialysis in traumatic brain injury and subarachnoid hemorrhage: state of the art. Neurocritical Care. 21 (1), 152-162 (2014).
  29. Amiridze, N., Dang, Y., Brown, O. R. Hydroxyl radicals detected via brain microdialysis in rats breathing air and during hyperbaric oxygen convulsions. Redox Report. 4 (4), 165-170 (1999).
  30. Chang, H. Y., Morrow, K., Bonacquisti, E., Zhang, W., Shah, D. K. Antibody pharmacokinetics in rat brain determined using microdialysis. MABS. 10 (6), 843-853 (2018).
  31. Wan, H. Y., et al. Pharmacokinetics of seven major active components of Mahuang decoction in rat blood and brain by LC-MS/MS coupled to microdialysis sampling. Naunyn-Schmiedeberg’s Archives of Pharmacology. 393 (8), 1559-1571 (2020).
  32. Zheng, H. Z., et al. Pharmacokinetic analysis of Huangqi Guizhi Wuwu decoction on blood and brain tissue in rats with normal and cerebral ischemia-reperfusion Injury by microdialysis with HPLC-MS/MS. Drug Design Development and Therapy. 14, 2877-2888 (2020).
  33. Bongaerts, J., et al. Sensitive targeted methods for brain metabolomic studies in microdialysis samples. Journal of Pharmaceutical and Biomedical Analysis. 161, 192-205 (2018).
  34. Zhang, Y. Q., Jiang, N., Yetisen, A. K. Brain neurochemical monitoring. Biosensors and Bioelectronics. 189, 113351 (2021).

Play Video

Citar este artigo
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).

View Video