利用体外大鼠脑切片研究变性主要事件的一种替代方法
Summary
我们提出了一种方法, 它可以进一步深入了解基础变性的早期事件, 并基于建立的前体脑技术, 结合体内和试管内实验的优点。此外, 它是一个独特的机会, 直接比较治疗和未经治疗的小组在同一解剖平面。
Abstract
尽管许多研究试图开发可靠的动物模型, 反映了变性的主要过程, 但很少有人被广泛接受。在这里, 我们提出了一个新的程序, 适应于已知的前体脑切片技术, 它提供了一个更接近的在体内类似的情况下, 比的试管准备, 以调查早期事件触发细胞变性, 作为观察到阿尔茨海默病 (AD)。这个变异包括简单和容易地可再生的步骤, 使保存选定的脑区域的解剖细胞构筑和它的地方功能在一个生理环境中。不同的解剖区域可以从同一大脑中获得, 提供了在一个站点、剂量和时间依赖性的方式下进行多项实验的机会。可能影响与此方法有关的结果的潜在限制与组织的保存有关,即,在切片和孵化步骤和剖面厚度期间保持其解剖完整性, 这能影响生化和免疫组化分析。这种方法可以用于不同的目的, 如探索分子机制参与生理或病理条件, 药物筛选, 或剂量反应化验。最后, 该议定书还可以减少在行为研究中使用的动物数量。在这里报告的应用最近首次被描述和测试的体外大鼠脑切片包含基底前脑 (BF), 这是一个主要影响的脑区的 AD. 具体地说, 它已经证明, 从乙酰胆碱酯酶 (疼痛) 的 C 终点产生的毒性肽的管理可能会提示一个 AD 样的轮廓, 触发, 沿 antero-后轴的 BF, 差异表达AD 中的蛋白质改变, 如 alpha7 烟碱受体 (α7 nAChR), 磷酸化头 (p 头) 和淀粉样蛋白β (Aβ)。
Introduction
AD 是一种慢性病理学特征的逐步神经退行性损伤影响不同的大脑区域, 如嗅皮质 (EC), BF, 海马 (HC), 和嗅觉灯泡 (OB)1,2,3, 4,5。AD 开发的后期阶段导致了逐渐的认知下降, 使这种疾病成为最常见的痴呆症, 大约占所有病例的 70%6。尽管人们广泛尝试了解导致 AD 的最初阶段, 但目前还没有一个明确的实验性指示来阐明它们。此外, 最流行的理论-“淀粉样假说”-越来越受到质疑, 因为它没有提供一个完整的形象解释广告病理, 也没有一个药物的目标已证明有效的7,8 ,9。
另一种被越来越关注的理论表明, 变性期间发生的初始机制与主要易受 AD3、10、11的神经元簇有关.,12,13,14. 这种异构的蜂窝中枢包含在 BF、中脑和脑干中, 项目到多个区域, 如 EC、HC 和 OB15,16。尽管它的神经元形态学和神经递质合成的多样性, 这一核心细胞在表达疼痛的共同特征, 也可以有一个非酶功能17,18。这种非经典的角色作为一个新的信号分子介导钙 (Ca2 +) 流入神经元, 可以接受营养或有毒事件有关 Ca2 +剂量, 可用性和神经元年龄17,18,19。
在变性期间, 观察到的细胞损失可能因此与这个非酶函数17,18,20, 这是归因于30mer 肽 (T30) 从疼痛 C 总站裂了20. 根据以前的结果, 进行细胞培养和光学成像18,21的准备工作, 我们通过一种新的方法, 通过一种基于前体大鼠脑切片的高炉结构, 表明 T30 诱导类似于 AD 的配置文件22。具体地说, 这种新的方法提供了比细胞培养更具生理的情况, 因为它保持了完整组织的许多特征, 从解剖到电路保存, 尽管时间窗口小时。我们应用这个协议来探索在变性的早期阶段发生的事件, 监测 T30 应用时的急性反应。
尽管大量的文献, 使用脑切片来调查隐含在神经损伤或神经发生的分子通路23,24, 此协议首次提供更直接和敏感的读出比较脊髓切片的常用用法。然而, 正如脊髓脑部分的情况一样, 这种急性切片程序也可以用于几个目的, 如评估神经保护或毒性分子, 发现在特定过程中发生的主要分子变化,中枢神经系统相关病理学的免疫组化分析和药理学测定。
Protocol
所有动物研究都是根据批准的议定书进行的。 注意: 在本节中, 提供了在实验过程中执行的主要阶段的顺序和建议的时间间隔 (图 1)。此外, 对该议定书的一步一步的描述由一个说明性的小组补充, 显示了在潜伏期后从脑切除到组织均匀化的关键行动 (图 2)。前面介绍了用于构建该设备的材料和说明的详细信息以及对世行分析的后续?…
Representative Results
这里提出的协议表明, 在含 BF 的部分 (图 3A) 中, 对有毒肽 T30 的管理, 以与站点相关的方式调节α7-nAChR、p 头和 Aβ的表达式。烟碱受体显示, 延髓处理的 hemislice 显著增加, 与它的控制对应 (切片 1, p = 0.0310) (图 3B), 而中间切片不显示任何变化的两个条件 (切片 2, p = 0.1195) (图 3B)。在后段中, T30-exp…
Discussion
该协议的主要方面是基于已建立良好的 “体外” 大脑技术, 允许同步测试两个镜面 hemislices, 从同一解剖平面获得, 监测其在应用特定条件 (控制或治疗);因此, 这提供了一个实验范式, 尽可能严密控制。如在神经退行性事件22中所见, 以时间、剂量和地点特定方式评估与神经元损伤有关的不同 neurochemicals 的可能性是一个基本特征。此方法将体内生理环境的优点与体外</…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作是由神经生物有限公司资助的。我们想感谢 Ferrati 博士和塞尔希奥·比埃拉·德梅洛博士圣乔瓦尼罗 (神经生物学) 对手稿的评论和建议。
Materials
| Sodium chloride (NaCl) | Sigma-Aldrich, Germany | S7653 | Reagent for aCSF preparation |
| Potassium chloride (KCl) | Sigma-Aldrich, Germany | P9333 | Reagent for aCSF preparation |
| Sodium bicarbonate (NaHCO3) | Sigma-Aldrich, Germany | S5761 | Reagent for aCSF preparation |
| Magnesium sulphate heptahydrate (MgSO4 (7H2O)) | Sigma-Aldrich, Germany | 63138 | Reagent for aCSF preparation |
| Potassium phosphate monobasic (KH2PO4) | Sigma-Aldrich, Germany | P5655 | Reagent for aCSF preparation |
| Hepes salt | Sigma-Aldrich, Germany | H7006 | Reagent for aCSF preparation |
| Hepes acid | Sigma-Aldrich, Germany | H3375 | Reagent for aCSF preparation |
| Glucose | Sigma-Aldrich, Germany | G7528 | Reagent for aCSF preparation |
| Calcium chloride dehydrate | Sigma-Aldrich, Germany | 223506 | Reagent for aCSF preparation |
| T30 peptide | Genosphere Biotechnologies, France | AChE-derived peptide tested | |
| Surgical dissecting kit | World Precision Instruments, USA | Item #: MOUSEKIT | Brain removal step |
| Surgical blades | Swann-Morton, UK | BS 2982 | Brain removal step |
| Filter paper | Fisher Scientific, USA | 11566873 | Brain preparation for slicing |
| Glue | Brain preparation for slicing | ||
| Vibratome | Leica, Germany | VT1000 S | Slicing |
| Brushes | Tissue handling | ||
| Oxygen canister | Sectioning and incubation phase | ||
| 1x Phosphate buffer saline (PBS) | Fisher Scientific, USA | BP2438-4 | Homogenization step |
| Phosphatase inhibitors | Fisher Scientific, USA | 1284-1650 | Homogenization step |
| Protease inhibitors | Roche complete PIC, USA | 4693116001 | Homogenization step |
| Pestles | Starlab, UK | I1415-5390 | Homogenization step |
| Microcentrifuge | |||
| Pierce 660 nm Protein Assay | Thermo Scientific, USA | 22660 | Protein concentration |
References
- Braak, H., Braak, E. Neuropathological stageing of Alzheimer-related changes. Acta Neuropathologica. 82 (4), 239-259 (1991).
- Schliebs, R. Basal forebrain cholinergic dysfunction in Alzheimer’s disease–interrelationship with beta-amyloid, inflammation and neurotrophin signaling. Neurochemical Research. 30 (6-7), 895-908 (2005).
- Schmitz, T. W., et al. Basal forebrain degeneration precedes and predicts the cortical spread of Alzheimer’s pathology. Nature Communications. 7, 13249 (2016).
- Fjell, A. M., McEvoy, L., Holland, D., Dale, A. M., Walhovd, K. B. What is normal in normal aging? Effects of aging, amyloid and Alzheimer’s disease on the cerebral cortex and the hippocampus. Progress in neurobiology. 117, 20-40 (2014).
- Kovács, T., Cairns, N. J., Lantos, P. L. Olfactory centres in Alzheimer’s disease: olfactory bulb is involved in early Braak’s stages. Neuroreport. 12 (2), 285-288 (2001).
- Winblad, B., et al. Defeating Alzheimer’s disease and other dementias: a priority for European science and society. The Lancet Neurology. 15 (5), 455-532 (2016).
- Herrup, K. The case for rejecting the amyloid cascade hypothesis. Nat Neurosci. 18 (6), 794-799 (2015).
- De Strooper, B., Karran, E. The Cellular Phase of Alzheimer’s Disease. Cell. 164 (4), 603-615 (2016).
- Scheltens, P., et al. Alzheimer’s disease. Lancet. 388 (10043), 505-517 (2016).
- Arendt, T., Brückner, M. K., Lange, M., Bigl, V. Changes in acetylcholinesterase and butyrylcholinesterase in Alzheimer’s disease resemble embryonic development-A study of molecular forms. Neurochemistry International. 21 (3), 381-396 (1992).
- Auld, D. S., Kornecook, T. J., Bastianetto, S., Quirion, R. Alzheimer’s disease and the basal forebrain cholinergic system: relations to β-amyloid peptides, cognition, and treatment strategies. Progress in Neurobiology. 68 (3), 209-245 (2002).
- Arendt, T., Bruckner, M. K., Morawski, M., Jager, C., Gertz, H. J. Early neurone loss in Alzheimer’s disease: cortical or subcortical?. Acta Neuropathol Commun. 3, 10 (2015).
- Mesulam, M. The Cholinergic Lesion of Alzheimer’s Disease: Pivotal Factor or The Cholinergic Lesion of Alzheimer’s Disease: Pivotal Factor or Side Show?. Learn Mem. , 43-49 (2004).
- Schliebs, R., Arendt, T. The cholinergic system in aging and neuronal degeneration. Behavioural Brain Research. 221 (2), 555-563 (2011).
- Mesulam, M. M., Mufson, E. J., Wainer, B. H., Levey, A. I. Central cholinergic pathways in the rat: An overview based on an alternative nomenclature (Ch1-Ch6). Neurosciences. 10 (4), 1185-1201 (1983).
- Mesulam, M., Mufson, E. J., Levey, A. I., Wainer, B. H. Cholinergic innervation of cortex by the basal forebrain: cytochemistry and cortical connections of the septal area, diagonal band nuclei, nucleus basalis (substantia innominata), and hypothalamus in the rhesus monkey. J Comp Neurol. 214 (2), 170-197 (1983).
- Greenfield, S. Discovering and targeting the basic mechanism of neurodegeneration: The role of peptides from the C-terminus of acetylcholinesterase: Non-hydrolytic effects of ache: The actions of peptides derived from the C-terminal and their relevance to neurodegenerat. Chemico-Biological Interactions. 203 (3), 543-546 (2013).
- Garcia-Ratés, S., et al. (I) Pharmacological profiling of a novel modulator of the α7 nicotinic receptor: Blockade of a toxic acetylcholinesterase-derived peptide increased in Alzheimer brains. Neuropharmacology. 105, 487-499 (2016).
- Eimerl, S., Schramm, M. The quantity of calcium that appears to induce neuronal death. Journal of neurochemistry. 62 (3), 1223-1226 (1994).
- Greenfield, S., Vaux, D. J. Commentary Parkinson’s Disease, Alzheimer’s Disease and Motor Neurone Disease: Identifying a Common Mechanism. Science. 113 (3), 485-492 (2002).
- Badin, A. S., Morrill, P., Devonshire, I. M., Greenfield, S. A. (II) Physiological profiling of an endogenous peptide in the basal forebrain: Age-related bioactivity and blockade with a novel modulator. Neuropharmacology. 105, 47-60 (2016).
- Brai, E., Stuart, S., Badin, A. -. S., Greenfield, S. A. A Novel Ex Vivo Model to Investigate the Underlying Mechanisms in Alzheimer’s Disease. Frontiers in Cellular Neuroscience. 11, 291 (2017).
- Cho, S., Wood, A., Bowlby, M. R. Brain slices as models for neurodegenerative disease and screening platforms to identify novel therapeutics. Current neuropharmacology. 5 (1), 19-33 (2007).
- Humpel, C. Organotypic brain slice cultures: A review. Neurosciences. 305, 86-98 (2015).
- Sakmann, B., Neher, E. Patch clamp techniques for studying ionic channels in excitable membranes. Annual review of physiology. 46, 455-472 (1984).
- Jensen, M. S., Lambert, J. D. C., Johansen, F. F. Electrophysiological recordings from rat hippocampus slices following in vivo brain ischemia. Brain Research. 554 (1-2), 166-175 (1991).
- Ferrati, G., Martini, F. J., Maravall, M. Presynaptic Adenosine Receptor-Mediated Regulation of Diverse Thalamocortical Short-Term Plasticity in the Mouse Whisker Pathway. Frontiers in Neural Circuits. 10, 1-9 (2016).
- Grinvald, A., Hildesheim, R. VSDI: a new era in functional imaging of cortical dynamics. Nature Reviews Neuroscience. 5 (11), 874-885 (2004).
- Badin, A. S., John, E., Susan, G. High-resolution spatio-temporal bioactivity of a novel peptide revealed by optical imaging in rat orbitofrontal cortex in vitro: Possible implications for neurodegenerative diseases. Neuropharmacology. 73, 10-18 (2013).
- Greenfield, S. A., Badin, A. S., Ferrati, G., Devonshire, I. M. Optical imaging of the rat brain suggests a previously missing link between top-down and bottom-up nervous system function. Neurophotonics. 4, 31213 (2017).
- Opitz-Araya, X., Barria, A. Organotypic hippocampal slice cultures. Journal of visualized experiments: JoVE. (48), (2011).
- Gong, C. -. X., Lidsky, T., Wegiel, J., Grundke-Iqbal, I., Iqbal, K. Metabolically active rat brain slices as a model to study the regulation of protein phosphorylation in mammalian brain. Brain Research Protocols. 6 (3), 134-140 (2001).
Citer Cet Article
Brai, E., Cogoni, A., Greenfield, S. A. An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices. J. Vis. Exp. (134), e57507, doi:10.3791/57507 (2018).