体内高分子量截离法从脑间质液中提取大细胞外蛋白的微透析方法
Summary
体内微透析已使大脑间质液中存在的分子从清醒的、自由行为的动物中收集。为了分析该化合物中相对较大的分子, 本文重点研究了用高分子量切断膜探针的微透析协议。
Abstract
在体内微透析是一种强大的技术, 收集从清醒, 自由行为的动物基于透析原理。虽然微透析是一种测量相对较小的分子, 包括氨基酸或神经递质的已建立的方法, 它最近也被用来评估大分子的动力学, 利用探针与高分子量切断膜。使用这种探针时, 微透析必须以推拉模式运行, 以避免探头内部积聚的压力。本文提供了分步协议, 包括立体定向手术, 以及如何建立微透析线收集蛋白质从。在微透析中, 药物可以系统地或直接输注到疫苗中。反向微透析技术是直接将化合物注入到其上的一种方法。在微透析灌注缓冲器中加入药物, 可使它们通过探针扩散到。以头蛋白为例, 通过对化学点燃癫痫的反向微透析, 揭示了其水平在刺激神经元活性方面的变化。本文结合其他体内方法, 介绍了微透析技术的优点和局限性。
Introduction
该方法包括总脑容积的 15-20%, 并提供了一个关键的微环境对信号传导, 基质运输和废物清除1。因此, 收集活体动物的能力将对各种生物过程和疾病机制带来更大的影响。体内微透析是从清醒的、自由移动的动物中取样和量化细胞外分子的为数不多的方法之一, 从而在神经科学研究领域2、3中成为有用的工具。在该方法中, 透膜的微透析探针插入脑中, 以相对缓慢的流速 (0.1-5 µL/分钟) 灌流灌注缓冲液。在此灌注过程中, 细胞外分子根据浓度梯度被动扩散到探针中, 并作为透析液收集。虽然本文的重点是对脑部标本的方法, 但无论是原则还是方法, 都可以通过适当的修改在必要的情况下应用于其他器官。
微透析在二十世纪六十年代代初首次被使用, 从那时起, 它被广泛用于收集小分子, 包括氨基酸或脑内神经递质。然而, 最近的商业可用性微透析探针与高分子量的切割膜 (100 kDa-3 MDa) 已扩大其应用到相对较大的蛋白质, 以及4,5,6 ,7。使用这些探针的研究结果发现, 长时间被认为是专属细胞质的头或α synuclein 的蛋白质也在生理上存在于4,5,8。
使用微透析探针与大型切断膜 (通常超过 1000 kDa) 的难点之一是, 由于探针中积聚的内压, 它们更容易受到超滤液的损耗。这里使用的微透析探针有一个独特的结构来避免这个问题。压力不会被建立由于这个结构, 因此微透析与这些探针应该操作在 “推挤-拉扯” 方式使用注射器泵灌注探针 (= 推挤) 和滚筒/蠕动泵收集从探针出口的透析器(= 拉)9 (虽然它需要推挤和拉扯泵, 由于压力取消透气孔存在于探针, 系统在技术上仅由拉扯泵驱动)。本文从引导套管植入的立体定向手术入手, 介绍了如何建立微透析线, 以便通过微透析探针与 1000 kDa 膜分离来采集。
Protocol
Representative Results
Discussion
高分子量切断膜的微透析必须采用推拉方式操作, 因此流速准确、恒定是至关重要的。流动速率的不准确性可能是气泡产生的原因和样品浓度不一致。如果流程不一致, 请检查所有连接是否有泄漏。如果问题仍然存在, 可能需要重新启动新的探头和油管。
Microdilaysis 探头通过灌注缓冲液不断灌注。因此, 细胞外分子没有足够的时间在灌注缓冲液中达到完全平衡。因此, 透析液中?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
这项工作得到了来自下个和年轻科学家 (B) (16K20969) 资助的 “对创新领域 (脑蛋白质老化和痴呆控制) (15H01552) 进行科学研究的资助” 的支持。作者感谢 David m. Holtzman 博士和 Cirrito 博士在这一方法的发展过程中提出的技术建议。
Materials
| The Univentor 820 Microsampler | Univentor | 8303002 | Refrigerated fraction collector |
| Syringe pump | KD scientific | KDS-101 | |
| Roller pump | Eicom microdialysis | ERP-10 | |
| Raturn Stand-Alone System | BASi | MD-1409 | Free-moving system |
| Dual species cage kit | BASi | CX-1600 | |
| AtmosLM Microdialysis probe (shaft length 8 mm, membrane length 2 mm) | Eicom microdialysis | PEP-8-02 | Shaft length for a probe, a guide, a dumy probe and a stereotaxic adaptor should be identical. |
| Microdialysis guide (shaft length 8 mm) | Eicom microdialysis | PEG-8 | |
| Microdialysis dummy probe (shaft length 8 mm) | Eicom microdialysis | PED-8 | |
| Bone screw | BASi | MD-1310 | |
| Super bond C&B set | Sunmedical | Dental cement | |
| Small animal Stereotaxic Instrument with digital display console | Kopf | Model 940 | Stereotaxic apparatus |
| Mouse and neonatal rat adaptor | Stoelting | 51625 | |
| Standard Ear Bars and Rubber Tips for Mouse Stereotaxic | Stoelting | 51648 | |
| Albumin solution from bovine serum | Sigma | A7284-50ML | 30% BSA solution |
| FEP tubing (70 cm) | Eicom microdialysis | JF-10-70 | Internal volume = 0.5 µL/cm |
| Teflon tubing (50 cm) | Eicom microdialysis | JT-10-50 | Internal volume = 0.08 µL/cm |
| Byton tube | Eicom microdialysis | JB-30 | |
| Intramedic luer stab adaptor 23G | BD | 427565 | Blunt end needle |
| Roller tube | Eicom microdialysis | RT-5S | Internal volume = 4 µL |
| Cap nut | Eicom microdialysis | AC-5 | |
| 0.25 mL microcentrifuge tube with cap | QSP | 503-Q | Tubes for fraction collector |
| Sterotaxic adaptor (shaft length 8 mm) | Eicom microdialysis | PESG-8 | |
| Connection needle | Eicom microdialysis | RTJ | |
| Mouse animal collar | BASi | MD-1365 | |
| High Speed Rotary Micromotor kit | FOREDOM | K.1070 | Drill |
| Picrotoxin | Sigma | P1675 | |
| Screw driver for bone screws | |||
| Scalpel | |||
| Cotton swab | |||
| Surgical clipper |
Referencias
- Lei, Y., Han, H., Yuan, F., Javeed, A., Zhao, Y. The brain interstitial system: Anatomy, modeling, in vivo measurement, and applications. Progress in Neurobiology. 157, 230-246 (2017).
- Kushikata, T., Hirota, K. Neuropeptide microdialysis in free-moving animals. Methods in Molecular Biology. 789, 261-269 (2011).
- Cirrito, J. R., et al. In vivo assessment of brain interstitial fluid with microdialysis reveals plaque-associated changes in amyloid-beta metabolism and half-life. The Journal of Neuroscience. 23 (26), 8844-8853 (2003).
- Emmanouilidou, E., et al. Assessment of α-synuclein secretion in mouse and human brain parenchyma. PLoS One. 6 (7), 1-9 (2011).
- Yamada, K., et al. In vivo microdialysis reveals age-dependent decrease of brain interstitial fluid tau levels in P301S human tau transgenic mice. The Journal of Neuroscience. 31 (37), 13110-13117 (2011).
- Ulrich, J. D., et al. In vivo measurement of apolipoprotein E from the brain interstitial fluid using microdialysis. Molecular Neurodegeneration. 8 (1), 13 (2013).
- Emmanouilidou, E., et al. GABA transmission via ATP-dependent K+ channels regulates α-synuclein secretion in mouse striatum. Brain. 139 (3), 871-890 (2016).
- Takeda, S., et al. Seed-competent high-molecular-weight tau species accumulates in the cerebrospinal fluid of Alzheimer’s disease mouse model and human patients. Annals of Neurology. 80 (3), 355-367 (2016).
- Yamada, K. In vivo Microdialysis of brain interstitial fluid for the determination of extracellular tau levels. Methods in Molecular Biology. 1523, 285-296 (2017).
- Kang, J. -. E., et al. Amyloid-β dynamics are regulated by orexin and the sleep-wake cycle. Science. 326 (November), 1005-1008 (2009).
- Cirrito, J. R., et al. Synaptic activity regulates interstitial fluid amyloid-beta levels in vivo. Neuron. 48 (6), 913-922 (2005).
- Bero, A. W., et al. Neuronal activity regulates the regional vulnerability to amyloid-β deposition. Nature Neuroscience. 14 (6), 750-756 (2011).
- Yamada, K., et al. Neuronal activity regulates extracellular tau in vivo. Journal of Experimental Medicine. 211 (3), 387-393 (2014).
- Pooler, A. M., Phillips, E. C., Lau, D. H. W., Noble, W., Hanger, D. P. Physiological release of endogenous tau is stimulated by neuronal activity. EMBO Reports. 14 (4), 389-394 (2013).
- Castellano, J. M., et al. Human apoE isoforms differentially regulate brain amyloid-β peptide clearance. Science Translational Medicine. 3 (89), 89ra57 (2011).
- Yamada, K., et al. Analysis of in vivo turnover of tau in a mouse model of tauopathy. Molecular Neurodegeneration. 10 (1), 55 (2015).
- Taylor, H., et al. Investigating local and long-range neuronal network dynamics by simultaneous optogenetics, reverse microdialysis and silicon probe recordings in vivo. Journal of Neuroscience Methods. 235, 83-91 (2014).
