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Introduction
Protocol
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神经科学

离体视网膜组织样品中线粒体呼吸和糖酵解的测定

Published: August 04, 2021
doi:
10.3791/62914
, ,
1Neurobiology, Neurodegeneration & Repair Laboratory, National Eye Institute,National Institutes of Health

Summary

这里描述的是使用商用生物分析仪在 离体 视网膜组织样品中进行线粒体应激测定和糖酵解速率测定的详细方案。

Abstract

线粒体呼吸是所有细胞中产生能量的关键途径，尤其是具有高度活跃代谢的视网膜光感受器。此外，光感受器还像癌细胞一样表现出高需氧糖酵解。这些代谢活动的精确测量可以为生理条件下和疾病状态下的细胞稳态提供有价值的见解。已经开发了基于微孔板的高通量测定法，用于测量活细胞中的线粒体呼吸和各种代谢活动。然而，其中绝大多数是为培养细胞开发的，尚未针对完整组织样品和 离体应用进行优化。这里描述的是详细的分步方案，使用基于微孔板的荧光技术，直接测量作为线粒体呼吸指标的耗氧率（OCR），以及细胞外酸化速率（ECAR）作为糖酵解的指标，在完整的 离体 视网膜组织中。该方法已被用于成功评估成年小鼠视网膜的代谢活动，并证明其在研究衰老和疾病的细胞机制中的应用。

Introduction

线粒体是必不可少的细胞器，通过协调多个关键的生理过程来调节细胞代谢、信号传导、稳态和细胞凋亡1。线粒体是细胞中通过氧化磷酸化（OXPHOS）产生三磷酸腺苷（ATP）的动力源，并提供支持几乎所有细胞事件的能量。大多数细胞氧在线粒体中代谢，在有氧呼吸过程中，它作为电子传递链（ETC）中的最终电子受体。细胞质基质中的糖酵解也可以产生低量的ATP，其中葡萄糖转化为丙酮酸盐，丙酮酸盐可以进一步转化为乳酸盐或转运到线粒体中并氧化成乙酰辅酶A，乙酰辅酶A是三羧酸循环（TCA循环）中的底物。

视网膜是哺乳动物中代谢活性最高的组织之一2，具有高水平的线粒体呼吸和极高的耗氧量3。视杆细胞和视锥体光感受器含有高密度的线粒体4，OXPHOS在视网膜中产生大多数ATP5。此外，视网膜还严重依赖需氧糖酵解6，7，通过将葡萄糖转化为乳酸5。线粒体缺陷与各种神经退行性疾病有关8，9;由于其独特的高能量需求，视网膜特别容易受到代谢缺陷的影响，包括影响线粒体OXPHOS4和糖酵解10的代谢缺陷。线粒体功能障碍和糖酵解缺陷与视网膜11、12 和黄斑13 退行性疾病、年龄相关性黄斑变性10、14、15、16 和糖尿病视网膜病变17、18 有关。因此，线粒体呼吸和糖酵解的准确测量可以为评估视网膜的完整性和健康状况提供重要参数。

线粒体呼吸可以通过测定耗氧率（OCR）来测量。鉴于葡萄糖转化为丙酮酸盐并随后转化为乳酸盐导致质子挤出到细胞外环境中并酸化，细胞外酸化速率（ECAR）的测量提供了糖酵解通量的指示。由于视网膜由具有亲密关系和积极协同作用的多种细胞类型组成，包括底物的交换6，因此必须在整个视网膜组织具有完整层压和电路的背景下分析线粒体功能和新陈代谢。在过去的几十年中，Clark型O2 电极和其他氧微电极已被用于测量视网膜中的氧气消耗19，20，21。这些氧电极在灵敏度、大样品体积要求以及需要连续搅拌悬浮样品方面具有重大局限性，这通常会导致细胞和组织环境的破坏。这里描述的方案是使用基于微孔板的荧光技术开发的，用于测量新鲜解剖 的离体 小鼠视网膜组织中的线粒体能量代谢。它允许使用 离体 视网膜组织的小样品（1 mm冲孔）同时对OCR和ECAR进行中等通量实时测量，同时避免悬浮和连续搅拌。

这里演示的是线粒体应激测定和糖酵解速率测定在新鲜解剖的视网膜打孔盘上的实验程序。该协议允许在 离体 组织环境中测量线粒体相关的代谢活动。与使用培养细胞进行的测定不同，此处获得的读数反映了组织水平上的组合能量代谢，并受到组织内不同细胞类型之间相互作用的影响。该协议从先前发布的版本22，23 进行了修改，以适应新一代带有胰岛捕获板的安捷伦海马细胞外通量24孔（XFe24）分析仪。检测培养基、注射化合物浓度和检测周期的次数/持续时间也针对视网膜组织进行了优化。给出了制备视网膜打孔盘的详细分步方案。有关程序设置和数据分析的更多信息可以从制造商的用户指南24，25，26获得。

Protocol

所有小鼠方案均由国家眼科研究所动物护理和使用委员会（NEI ASP# 650）批准。将小鼠饲养在12小时的明暗条件下，并按照《实验动物护理和使用指南》、实验动物资源研究所和《关于实验动物人道护理和使用的公共卫生服务政策》的建议进行护理。 1. 补水传感器墨盒及测定介质的制备 在实验前一天，向实用板的每个孔中加入1mL校准培养基。将液压助推器盖放在顶部，?…

Representative Results

这里报告的数据是具有代表性的线粒体应激测定，显示OCR痕量（图1）和糖酵解速率测定显示OCR痕量和ECAR痕量（图2），这是使用来自4个月大转基因 Nrl-L-EGFP 小鼠36 （C57B / L6背景）的新鲜解剖的1mm视网膜打孔盘进行的。这些小鼠在视杆光感受器中特异性表达GFP，而不改变正常的视网膜发育，组织学和生理学，并已被广泛用作视网…

Discussion

此处提供了使用离体，新鲜解剖的视网膜打孔盘进行基于微孔板的线粒体呼吸和糖酵解活性测定的详细说明。该方案已优化为：1）确保对离体视网膜组织使用合适的测定介质;2）采用适当尺寸的视网膜打孔盘，以获得落在机器最佳检测范围内的OCR和ECAR读数;3）涂层网嵌件，增强视网膜冲头的粘合性，在测量周期内读数稳定;4）使用最佳浓度的每种注射药物化合物;5）确保改变的周期长…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项工作得到了国家眼科研究所壁内研究计划（ZIAEY000450和ZIAEY000546）的支持。

Materials

1X PBS Thermo Fisher 14190-144
2-Deoxy glucose (2-DG), 500 mM stock solution Sigma D6134 Dissolve in Seahorse XF DMEM medium, prepare ahead of time
30-gauge needle BD Precision Glide 305106
Antimycin A, 10 mM stock solution Sigma A8674 Dissolve in DMSO, prepare ahead of time
Bam15, 10 mM stock solution TimTec ST056388 Dissolve in DMSO, prepare ahead of time
Biopsy puncher, 1 mm Integra Miltex 33-31AA
Cell-Tak Corning Life Sciences CB40240
CO2 asphyxiation chamber
Dissection forceps-Dumont #5 Fine Science Tools 11251-10 Stright tip
Dissection forceps-Dumont #7 Fine Science Tools 11274-20 Curved tip
Dissection microscope
DMSO Sigma D2438
Graefe forceps Fine Science Tools 11051-10 Curved, Serrated tip
Microscissors Fine Science Tools 15004-08 Curved tip
NaOH solution, 1 M Sigma-Aldrich S8263 Aqueous solution, prepare ahead of time
Rotenone, 10 mM stock solution Sigma R8875 Dissolve in DMSO, prepare ahead of time
Seahorse calibration medium Agilent 100840-000
Seahorse XF 1.0 M glucose Agilent 103577-100
Seahorse XF 100 mM pyruvate Agilent 103578-100
Seahorse XF 200 mM glutamine Agilent 103579-100
Seahorse XF DMEM medium Agilent 103575-100 pH 7.4, with 5 mM HEPES
Seahorse XFe24 Islet Capture FluxPak Agilent 103518-100 Containing Sensor Cartridge and Islet Capture microplate
Seahorse XFe24, Extra Cellular Flux Analyzer Agilent
Sodium bicarbonate solution, 0.1 M Sigma-Aldrich S5761 Aqueous solution, prepare ahead of time
Superfine eyelash brush Ted Pella 113
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References

  1. Nunnari, J., Suomalainen, A. Mitochondria: In sickness and in health. Cell. 148 (6), 1145-1159 (2012).
  2. Wong-Riley, M. T. Energy metabolism of the visual system. Eye Brain. 2, 99-116 (2010).
  3. Yu, D. Y., Cringle, S. J. Oxygen distribution and consumption within the retina in vascularised and avascular retinas and in animal models of retinal disease. Progress in Retina and Eye Research. 20, 175-208 (2001).
  4. Barot, M., Gokulgandhi, M. R., Mitra, A. K. Mitochondrial dysfunction in retinal diseases. Current Eye Research. 36 (12), 1069-1077 (2011).
  5. Joyal, J. S., Gantner, M. L., Smith, L. E. H. Retinal energy demands control vascular supply of the retina in development and disease: The role of neuronal lipid and glucose metabolism. Progress in Retina and Eye Research. 64, 131-156 (2018).
  6. Hurley, J. B., Lindsay, K. J., Du, J. Glucose, lactate, and shuttling of metabolites in vertebrate retinas. Journal of Neuroscience Research. 93 (7), 1079-1092 (2015).
  7. Haydinger, C. D., Kittipassorn, T., Peet, D. J. Power to see-Drivers of aerobic glycolysis in the mammalian retina: A review. Clinical and Experimental Ophthalmology. 48 (8), 1057-1071 (2020).
  8. Wright, A. F., et al. Lifespan and mitochondrial control of neurodegeneration. Nature Genetics. 36, 1153-1158 (2004).
  9. Bossy-Wetzel, E., Schwarzenbacher, R., Lipton, S. A. Molecular pathways to neurodegeneration. Nature Medicine. 10, 2-9 (2004).
  10. Leveillard, T., Philp, N. J., Sennlaub, F. Is retinal metabolic dysfunction at the center of the pathogenesis of age-related macular degeneration. International Journal of Molecular Sciences. 20 (3), (2019).
  11. Vlachantoni, D., et al. Evidence of severe mitochondrial oxidative stress and a protective effect of low oxygen in mouse models of inherited photoreceptor degeneration. Human Molecular Genetics. 20 (2), 322-335 (2011).
  12. Grenell, A., et al. Loss of MPC1 reprograms retinal metabolism to impair visual function. Proceedings of the National Academy of Science U. S. A. 116 (9), 3530-3535 (2019).
  13. Wright, A. F., Chakarova, C. F., Abd El-Aziz, M. M., Bhattacharya, S. S. Photoreceptor degeneration: genetic and mechanistic dissection of a complex trait. Nature Reviews in Genetics. 11 (4), 273-284 (2010).
  14. Jarrett, S. G., Boulton, M. E. Consequences of oxidative stress in age-related macular degeneration. Molecular Aspects of Medicine. 33 (4), 399-417 (2012).
  15. Rozing, M., et al. Age-related macular degeneration: A two-level model hypothesis. Progress in Retina Eye Research. 76, 100825 (2020).
  16. Yokosako, K., et al. Glycolysis in patients with age-related macular degeneration. Open Ophthalmology Journal. 8, 39-47 (2014).
  17. Bek, T. Mitochondrial dysfunction and diabetic retinopathy. Mitochondrion. 36, 4-6 (2017).
  18. Yumnamcha, T., Guerra, M., Singh, L. P., Ibrahim, A. S. Metabolic dysregulation and neurovascular dysfunction in diabetic retinopathy. Antioxidants. 9 (12), (2020).
  19. Futterman, S., Kinoshita, J. H. Metabolism of the retina. I. Respiration of cattle retina. Journal of Biological Chemistry. 234 (4), 723-726 (1959).
  20. Linsenmeier, R. A. Effects of light and darkness on oxygen distribution and consumption in the cat retina. Journal of General Physiology. 88 (4), 521-542 (1986).
  21. Medrano, C. J., Fox, D. A. Oxygen consumption in the rat outer and inner retina: light- and pharmacologically-induced inhibition. Experiments in Eye Research. 61 (3), 273-284 (1995).
  22. Kooragayala, K. Quantification of oxygen consumption in retina ex vivo demonstrates limited reserve capacity of photoreceptor mitochondria. Investigative Ophthalmology and Visual Science. 56 (13), 8428-8436 (2015).
  23. Adlakha, Y. K., Swaroop, A. Determination of mitochondrial oxygen consumption in the retina ex vivo: applications for retinal disease. Methods in Molecular Biology. 1753, 167-177 (2018).
  24. . Agilent Mitocondrial stress test user guide Available from: https://www.agilent.com/cs/library/usermanuals/public/XF_Cell_Mito_Stress_Test_Kit_User_Guide.pdf (2021)
  25. . Agilent Glycolytic rate assay user guide Available from: https://www.agilent.com/cs/library/usermanuals/public/103344-400.pdf (2021)
  26. . Agilent wave 2.6 user guide Available from: https://www.agilent.com/cs/library/usermanuals/public/103344-400.pdf (2021)
  27. . AVMA Guidelines for the Euthanasia of Animals Available from: https://www.avma.org/sites/default/files/2020-01/2020-Euthanasia-Final-1-17-20.pdf (2021)
  28. . Improving Quantification of Cellular Glycolytic Rate Using Agilent Seahorse XF Technology Available from: https://www.agilent.com/cs/library/whitepaper/public/whitepaper-improve-quantification-of-cellular-glycolytic-rate-cell-analysis-5991-7894en-agilent.pdf (2021)
  29. . Report Generator User Guide Agilent Seahorse XF Cell Mito Stress Test Available from: https://www.agilent.com/cs/library/usermanuals/public/Report_Generator_User_Guide_Seahorse_XF_Cell_Mito_Stress_Test_Single_File.pdf (2021)
  30. . Agilent Seahorse XF Buffer Factor Protocol Available from: https://www.agilent.com/cs/library/usermanuals/public/usermanual-xf-buffer-factor-protocol-cell-analysis-S7888-10010en-agilent.pdf (2021)
  31. . Agilent sensor cartridges and cell culture microplates Available from: https://www.agilent.com/cs/library/brochures/5991-8657EN_seahorse_plastics_brochure.pdf (2021)
  32. . Agilent Seahorse XF Glycolysis Stress Test Kit User Guide Available from: https://www.agilent.com/cs/library/usermanuals/public/XF_Glycolysis_Stress_Test_Kit_User_Guide.pdf (2021)
  33. Fan, Y. Y. A bioassay to measure energy metabolism in mouse colonic crypts, organoids, and sorted stem cells. American Journal of Physiology-Gastrointestinal and Liver Physiology. 309, 1-9 (2015).
  34. Huang, L., et al. Ductal pancreatic cancer modeling and drug screening using human pluripotent stem cell- and patient-derived tumor organoids. Nature Medicine. 21 (11), 1364-1371 (2015).
  35. Jeon, C. J., Strettoi, E., Masland, R. H. The major cell populations of the mouse retina. Journal of Neuroscience. 18 (21), 8936-8946 (1998).
  36. Akimoto, M., et al. Targeting of GFP to newborn rods by Nrl promoter and temporal expression profiling of flow-sorted photoreceptors. Proceedings of the National Academy of Science U. S. A. 103 (10), 3890-3895 (2006).
  37. Kenwood, B. M., et al. Identification of a novel mitochondrial uncoupler that does not depolarize the plasma membrane. Molecular Metabolism. 3 (2), 114-123 (2014).
  38. Corso-Diaz, X., et al. Genome-wide profiling identifies DNA methylation signatures of aging in rod photoreceptors associated with alterations in energy metabolism. Cell Reports. 31 (3), 107525 (2020).
  39. Berkowitz, B. A., et al. Mitochondrial respiration in outer retina contributes to light-evoked increase in hydration in vivo. Investigative Ophthalmology and Visual Science. 59 (15), 5957-5964 (2018).
  40. Joyal, J. S., et al. Retinal lipid and glucose metabolism dictates angiogenesis through the lipid sensor Ffar1. Nature Medicine. 22 (4), 439-445 (2016).

Tags

Keywords: Mitochondrial RespirationGlycolysisEx VivoSeahorse AnalysisInherited Retinal DegenerationRetinal DissectionMouse RetinaFluorophore ActivationAssay MediumRetinal Punch Preparation
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Jiang, K., Nellissery, J., Swaroop, A. Determination of Mitochondrial Respiration and Glycolysis in Ex Vivo Retinal Tissue Samples. J. Vis. Exp. (174), e62914, doi:10.3791/62914 (2021).
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