激光诱导的斑马鱼视网膜变性及再生模型中的 m ü ller 胶质细胞活化
Summary
斑马鱼是研究脊椎动物视网膜变性/再生机制的流行动物模型。本议定书描述一种方法, 以诱导局部损伤破坏外视网膜, 对内视网膜的损害最小。随后, 我们监测在体内视网膜形态学和 m ü ller 胶质细胞反应在整个视网膜再生。
Abstract
硬和哺乳动物之间的一个奇妙的区别是硬视网膜在严重损伤后的视网膜神经形成和再生的终身潜能。调查斑马鱼的再生途径可能带来新的见解, 以制定创新的策略, 治疗视网膜退行性疾病的哺乳动物。在此, 我们的重点是通过 532 nm 半导体激光器诱导成人斑马鱼外侧视网膜病灶。局部损伤允许对视网膜变性和在损伤区域直接再生的生物过程进行调查。使用无创光学相干断层扫描 (OCT), 我们能够定义受损区域的位置, 并监测后续再生在体内.事实上, OCT 成像产生的高分辨率, 横断面图像的斑马鱼视网膜, 提供的信息, 以前只提供了组织学分析。为了证实 real-time OCT 的数据, 通过免疫组织化学研究视网膜损伤诱导后的组织学切片和再生反应。
Introduction
视力可能是人类最基本的感觉, 它的损害具有很高的社会经济影响。在工业化世界中, 视网膜退行性疾病占成年人群视力丧失和失明的大多数1。视网膜色素变性 (RP) 是最常见的遗传性失明的原因, 在人的年龄之间的20和 60, 影响约150万人在全球范围内2,3。这是一个异构家族遗传性视网膜疾病的特点是逐步丧失的光 (减贫) 后, 视网膜色素上皮变性, 随后, 胶质和重塑内神经元4。疾病的过程可以解释的两个 PR 细胞类型的增量损失, 通常从棒开始, 这是负责的消色差视觉在昏暗的光和锥, 这是必不可少的颜色视觉和视力5。单个基因缺陷足以引起 RP。到目前为止, 超过45基因的130突变已经与疾病6相关。这导致不同的疾病表型, 是一个原因, 基因治疗是 non-generalizable, 从而一个复杂的治疗方法。因此, 迫切需要制定新的一般治疗方法来治疗致盲疾病的视网膜退化。
视网膜变性常涉及 PR 损失;因此, PR 细胞死亡是视网膜退行性过程的标志7。它已经证明, PR 细胞死亡刺激 m ü ller 胶质细胞 (MC) 激活和增殖8。MCs, 在脊椎动物视网膜的主要胶质细胞类型, 曾经被认为只是一个 “胶水” 之间的视网膜神经元。近年来, 许多研究表明, MCs 的作用不仅仅是结构支持9。在不同的功能中, MCs 也参与神经再生和修复10。事实上, 在对变性视网膜的扩散因素的反应中, MCs 显著增加胶质纤维酸性蛋白的表达。因此, 在视网膜损伤和变性11中, 胶质蛋白标签可以作为 MC 活化的标志。
最近, 我们开发了一种新的局灶损伤的适应, 利用激光诱导斑马鱼视网膜变性 (斑马鱼)。局灶性损伤有利于研究某些生物学过程, 如细胞迁移到受伤部位, 以及视网膜再生过程中发生的事件的准确时间12。此外, 由于其视觉系统与其他脊椎动物的相似性, 斑马鱼在视觉研究中已经变得非常重要。人和硬视网膜的大体形态和组织学特征显示出很少的差异。因此, 人类和斑马鱼视网膜包含相同的细胞类组织在同一层模式, 其中感光光占据最外层, 而视网膜投射神经元, 神经节细胞, 居住在最里面神经元层, 近于晶状体。视网膜神经元, 突, 两极, 和水平细胞, 定位在感光细胞和神经节单元层之间13。此外, 斑马鱼视网膜是锥体控制, 因此更接近人的视网膜比, 例如, 密集研究的啮齿动物视网膜。硬和哺乳动物之间的一个奇妙的区别是鱼类视网膜的持续神经再生和损伤后的视网膜重建。在斑马鱼, MCs 能分化和斡旋再生在受伤的视网膜14,15。在鸡肉中, MCs 也有能力重新进入细胞周期和分化。在成年鱼类视网膜损伤后, MCs 采用了祖细胞和干细胞的某些特征, 迁移到受损的视网膜组织, 产生新的神经元16。哺乳动物 mcs 的基因表达谱显示出与视网膜祖细胞有意想不到的相似性, 而在鸡、啮齿动物甚至人类视网膜中, mcs 的内在神经源潜能的证据也在增长17。然而, 为什么鸟类和哺乳动物的再生反应比起鱼类的健壮反应还要低呢？因此, 了解斑马鱼内源性修复机制可能会提出刺激哺乳动物和人类视网膜再生的策略。采用 MCs 内源性修复机制作为治疗视网膜变性患者的治疗工具, 将对我们的社会产生显著的影响。
在此, 我们提供了在眼科研究中使用变性/再生模型的必要步骤。我们首先关注的是诱导感觉神经视网膜的病灶损伤, 然后是在损伤部位发生的事件的成像, 最后是对相邻 MCs 的参与。一般的协议是相对容易执行, 并打开了各种可能性, 评估视网膜后。
Protocol
Representative Results
Discussion
通过毒素介导的细胞死亡22、机械损伤23和热损伤24等不同方法对斑马鱼视网膜再生/变性进行了研究。我们使用了 532 nm 二极管激光器来破坏斑马鱼视网膜。因此, 我们的模型提供了几个优点。例如, 我们迅速建立了一个明确界定的损伤区域, 在外部视网膜, 特别是在减贫战略层。此外, 这种实验装置可以被修改, 以产生较大的损害区域, 以研究其?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
我们感谢马丁 Zinkernagel, MD, 博士和仪 Reisenhofer, 博士为她的科学投入建立模型和费德里卡比西纳尼为她出色的技术援助。
Materials
| Acid hematoxylin solution | Sigma-Aldrich, Buchs, Switzerland | 2852 | |
| Albumin | Sigma-Aldrich, Buchs, Switzerland | A07030 | |
| Bovine serum albumin (BSA) | Sigma-Aldrich, Buchs, Switzerland | 5470 | |
| Dako Pen | Dako, Glostrup, Danmark | S2002 | |
| DAPI mounting medium | Vector Labs, Burlingame, CA, USA | H-1200 | |
| Eosin G aqueous solution 0.5% | Carl Roth, Arlesheim, Switzerland | X883.2 | |
| Ethanol | Sigma-Aldrich, Buchs, Switzerland | 2860 | |
| Ethylene diamine tetraacetic acid (EDTA) | Sigma-Aldrich, Buchs, Switzerland | ED | |
| Eukitt | Sigma-Aldrich, Buchs, Switzerland | 3989 | |
| Goat anti-rabbit IgG H&L Alexa Fluor® 488 | Life Technologies, Zug, Switzerland | A11008 | |
| Goat anti-mouse IgG H&L Alexa Fluor® 594 | Life Technologies, Zug, Switzerland | A11020 | |
| Goat normal serum | Dako, Glostrup, Danmark | X0907 | |
| Hydrogel contact lens | Johnson & Johnson AG, Zug, Switzerland | n.a. | 1-Day Acuvue Moist |
| Hydroxypropylmethylcellulose 2% | OmniVision, Neuhausen, Switzerland | n.a. | Methocel 2% |
| Ethyl 3-aminobenzoate methanesulfonate | Sigma-Aldrich, Buchs, Switzerland | A5040 | Tricaine, MS-222 |
| Visulas 532s | Carl Zeiss Meditec AG, Oberkochen, Germany | n.a. | 532 nm laser |
| Mouse anti-GS monoclonal antibody | Millipore, Billerica, MA, USA | MAB302 | |
| HRA + OCT Imaging System | Heidelberg Engineering, Heidelberg, Germany | n.a. | Spectralis |
| Heidelberg Eye Explorer | Heidelberg Engineering, Heidelberg, Germany | n.a. | Version 1.9.10.0 |
| Paraformaldehyde (PFA) | Sigma-Aldrich, Buchs, Switzerland | P5368 | |
| Phosphate buffered saline (PBS) | Sigma-Aldrich, Buchs, Switzerland | P5368 | |
| Rabbit anti-GFAP polyclonal antibody | Invitrogen, Waltham, MA, USA | 180063 | |
| Silicone pin holder | Huco Vision AG Switzerland | n.a. | Cut by hand from silicone pin mat of the sterilization tray accordingly. |
| Slit lamp BM900 | Haag-Streit AG, Koeniz, Switzerland | n.a. | |
| Slit lamp adapter | Iridex Corp., Mountain View, CA, USA | n.a. | |
| Superfrost Plus glass slides | Gehard Menzel GmbH, Braunschweig, Germany | 10149870 | |
| TgBAC (gfap:gfap-GFP) zf167 (AB) strain | KIT, Karlsruhe, Germany | 15204 | http://zfin.org/ZDB-ALT-100308-3 |
| Tris buffered saline (TBS) | Sigma-Aldrich, Buchs, Switzerland | P5912 | |
| Tween 20 | Sigma-Aldrich, Buchs, Switzerland | P1379 | |
| 78D non-contact slit lamp lens | Volk Optical, Mentor, OH, USA | V78C | |
| Xylene | Sigma-Aldrich, Buchs, Switzerland | 534056 | |
| Ocular fundus laser lens | Ocular Instruments, Bellevue, WA, USA | OFA2-0 | |
| 2100 Retriever | Aptum Biologics Ltd., Southampton, United Kingdom | R2100-EU | Steamer |
Riferimenti
- Haddad, S., Chen, C. A., Santangelo, S. L., Seddon, J. M. The genetics of age-related macular degeneration: a review of progress to date. Surv. Ophthalmol. 51 (4), 316-363 (2006).
- Stefano Ferrari, S., Di Iorio, E., Barbaro, V., Ponzin, D., Sorrentino, F. S., Parmeggiani, F. Retinitis Pigmentosa: Genes and Disease Mechanisms. Curr Genomics. 12 (4), 238-249 (2011).
- Berson, E. L. Retinitis pigmentosa. The Friedenwald Lecture. Invest Ophthalmol Vis Sci. 34 (5), 1659-1676 (1993).
- Strettoi, E. A Survey of Retinal Remodeling. Front Cell Neurosci. 9, 494 (2015).
- Hartong, D. T., Berson, E. L., Dryja, T. P. Retinitis pigmentosa. Lancet. 368, 1795-1809 (2006).
- Wang, D. Y., Chan, W. M., Tam, P. O., Baum, L., Lam, D. S., Chong, K. K., Fan, B. J., Pang, C. P. Gene mutations in retinitis pigmentosa and their clinical implications. Clin Chim Acta. 351 (1-2), 5-16 (2005).
- Pierce, E. A. Pathways to photoreceptor cell death in inherited retinal degenerations. BioEssays. 23, 605-618 (2001).
- Tackenberg, M. A., Tucker, B. A., Swift, J. S., Jiang, C., Redenti, S., Greenberg, K. P., Flannery, J. G., Reichenbach, A., Young, M. J. Muller cell activation, proliferation and migration following laser injury. Mol. Vis. , 1886-1896 (2009).
- Newman, E., Reichenbach, A. The Müller cell: a functional element of the retina. Trends Neurosci. 19 (8), 307-312 (1996).
- Kubota, R., Hokoc, J. N., Moshiri, A., McGuire, C., Reh, T. A. A comparative study of neurogenesis in the retinal ciliary marginal zone of homeothermic vertebrates. Brain Res Dev Brain Res. 134, 31-41 (2002).
- Zhao, T. T., Tian, C. Y., Yin, Z. Q. Activation of Müller cells occurs during retinal degeneration in RCS rats. Adv Exp Med Biol. 664, 575-583 (2010).
- DiCicco, R. M., Bell, B. A., Kaul, C., Hollyfield, J. G., Anand-Apte, B., Perkins, B. D., Tao, Y. K., Yuan, A. Retinal Regeneration Following OCT-Guided Laser Injury in Zebrafish. Invest Ophthalmol Vis Sci. 55 (10), 6281-6288 (2014).
- Bilotta, J., Saszik, S. The zebrafish as a model visual system. Int. J. Dev. Neurosci. , 621-629 (2001).
- Fausett, B. V., Goldman, D. A role for alpha1 tubulin-expressing Müller glia in regeneration of the injured zebrafish retina. J Neurosci. 26 (23), 6303-6313 (2006).
- Yurco, P., Cameron, D. A. Responses of Müller glia to retinal injury in adult zebrafish. Vision Res. 45, 991-1002 (2005).
- Ashutosh, P. J., Roesch, K., Cepko, C. L. Development and neurogenic potential of Müller gial cells in the vertebrate retina. Prog Retin Eye Res. 28 (4), 249-262 (2009).
- Xia, X., Ahmad, I. Unlocking the Neurogenic Potential of Mammalian Müller Glia. Int J Stem Cells. 9 (2), 169-175 (2016).
- Brand, M., Granato, M., Nüsslein-Volhard, C., Nüsslein-Volhard, C., Dahm, R. Keeping and raising zebrafish. Zebrafish: A Practical Approach. , 7-38 (2002).
- Riepe, R. E., Norenburg, M. D. Müller cell localisation of glutamine synthetase in rat retina. Nature. 268 (5621), 654-655 (1977).
- Derouiche, A., Rauen, T. Coincidence of L-glutamate/L-aspartate transporter (GLAST) and glutamine synthetase (GS) immunoreactions in retinal glia: evidence for coupling of GLAST and GS in transmitter clearance. J Neurosci Res. 42 (1), 131-143 (1995).
- Bignami, A., Dahl, D. The radial glia of Müller in the rat retina and their response to injury. An immunofluorescence study with antibodies to the glial fibrillary acidic (GFA) protein. Exp Eye Res. 28 (1), 63-69 (1979).
- Sherpa, T., Fimbel, S. M., Mallory, D. E., Maaswinkel, H., Spritzer, S. D., Sand, J. A., Li, L., Hyde, D. R., Stenkamp, D. L. Ganglion cell regeneration following whole-retina destruction in zebrafish. Dev Neurobiol. 68 (2), 166-181 (2008).
- Cameron, D. A., Carney, L. H. Cell mosaic patterns in the native and regenerated inner retina of zebrafish: implications for retinal assembly. J Comp Neurol. 416 (3), 356-367 (2000).
- Raymond, P. A., Barthel, L. K., Bernardos, R. L., Perkowski, J. J. Molecular characterization of retinal stem cells and their niches in adult zebrafish. BMC Dev Biol. 6, 36 (2006).
- Bailey, T. J., Davis, D. H., Vance, J. E., Hyde, D. R. Spectral-domain optical coherence tomography as a noninvasive method to assess damaged and regenerating adult zebrafish retinas. Invest Ophthalmol Vis Sci. 53 (6), 3126-3138 (2012).
- Koinzer, S., Saeger, M., Hesse, C., Portz, L., Kleemann, S., Schlott, K., Brinkmann, R., Roider, J. Correlation with OCT and histology of photocoagulation lesions in patients and rabbits. Acta Ophthalmol. 91 (8), e603-e611 (2013).
- Wan, J., Zheng, H., Chen, Z. L., Xiao, H. L., Shen, Z. J., Zhou, G. M. Preferential regeneration of photoreceptor from Müller glia after retinal degeneration in adult rat. Vision Res. (2), 223-234 (2008).
- Thomas, J. L., Thummel, R. A novel light damage paradigm for use in retinal regeneration studies in adult zebrafish. J Vis Exp. (80), e51017 (2013).
