表征的分子机制<em在体内</em>紫外线辐射诱发白内障
Summary
白内障是导致失明的世界。太阳紫外线辐射（UVR）是白内障发展的主要危险因素。远紫外线B诱导白内障动物模型的开发。在这篇文章中，我们描述了白内障的形成进行调查：暴露于紫外线辐射，定量RT-PCR和免疫组化的方法。
Abstract
白内障是导致失明的世界1。世界卫生组织定义白内障混浊的眼睛，阻碍了光传输的镜头。白内障是一种多因素的疾病，与糖尿病，吸烟，紫外线辐射（UVR），酒精，电离辐射，类固醇和高血压有关。有强烈的实验2-4和流行病学证据5,6，紫外线辐射会导致白内障。我们开发了两种麻醉和非麻醉动物的紫外线B诱导白内障的动物模型。
唯一的治疗白内障的手术治疗，但这种治疗方法是无法访问的所有。据估计10年白内障的发病延迟可以减少白内障手术的需要的50％9。为了延缓白内障的发生率，它是需要了解白内障的形成机制和有效的预防地层几种学习策略。白内障发展的机制之一，细胞凋亡起着至关重要的作用在启动白内障在人类和动物的10。最近我们的重点是在镜头凋亡的机制白内障的发展8,11,12。据预测，更好地了解紫外线辐射的影响，对细胞凋亡通路的发现新的药物，以防止白内障的可能性。
在这篇文章中，我们将介绍如何白内障可以通过实验诱导体内暴露于UVR-B。进一步的RT-PCR和免疫组化被作为工具来研究UVR-B诱发白内障的分子机制。
Protocol
1。暴露于紫外线辐射前15分钟的曝光，麻醉一个雌性Sprague-Dawley大鼠用90毫克/千克Ketalar（氯胺酮）和10毫克/公斤Rompun（甲苯噻嗪）通过腹膜内注射的混合物。 将动物在大鼠阻挡，并拧紧而不会造成一个中继线挤压13，直到固定大鼠的皮带。 灌输Mydriacyl（托），10毫克/毫升，在两只眼睛的大鼠诱导散瞳。 将动物，使一只眼睛被靠住一个狭窄的光束的紫外线辐射?…
Representative Results
测量中的变化的各种来源，估计与方差分析，并发现，考虑三个测量每只动物的方差用于测量的时间是在15％的动物的顺序。因此，考虑到整个透镜分析，这是不可能的，以增加的精度。正交试验阐明了统计上显着的对比度，120小时之间延迟的时间间隔与较短的延迟时间间隔的caspase-3的消息。 在体内紫外线辐射暴露诱导的caspase-3的表达。 <img alt="?…
Discussion
本文介绍的方法，使学习在UVR-B引起的白内障发生的分子事件。
考虑，大多数信息可用于在体内的紫外线辐射性白内障是来自实验白化只SD大鼠7日，16日，17日，18日，19后，我们决定在目前的研究中使用的白化SD大鼠。年龄的影响是6周大的。的性别是女性，因为在对男性相比，女性较少有过敏尿。此外，有没有性别差异的严重程度的紫外线辐射性白内障…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
这项工作是支持的卡罗林斯卡研究所的KID资金，瑞典辐射保护局，卡罗林斯卡医学院眼科研究基金会，Konung古斯塔夫枪OCH贝蒂尔Stohnes Stiftelse，圣Erik的眼科医院研究基金会，Ögonfonden：秒OCH Drottning Viktorias Frimurarstiftelse。
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
| Ketalar | Pfizer | 150086 | 50 mg/ml |
| Rompun | Bayer | 022545 | 20 mg/ml |
| Oculentum Simplex | Apoteket, Sweden | 336164 | 5 g |
| Mydriacyl | Alcon | 00352 | 10 mg/ml |
| Balanced salt solution | Alcon | 0007950055 | |
| 3.5 ul β-mercaptoethanol | |||
| NucleoSpin RNA II total RNA isolation kit | Macherey-Nagel GmbH&Co, Duren, Germany | 740955.50 | |
| p53 DNA specific primers | biomers.net GmbH | Custom made | |
| Taq DNA polymerase, dNTPack | Roche | 04 728 866 001 | |
| Agarose | Sigma | A5093 | |
| Ethidium bromide solution 0.5 mg/ml | Sigma | E1385 | |
| Nano-Drop ND-1000 spectrophotometer | NanoDrop Products | ||
| 1st Strand cDNA synthesis kit for RT-PCR (AMV) | Roche Diagnostics GmbH | 11 483 188 001 | |
| iCycler MyiQ Single Color Real Time PCR detection system | Bio-Rad Laboratories | ||
| 96-well plate | Sarstedt | 72.1979.202 | |
| TaqMan Gene Expression Master Mix | Applied Biosystems | 4369016 | |
| TaqMan Gene Expression Assay for caspase 3 | Applied Biosystems | Rn00563902_m1 | |
| TaqMan Gene Expression Assay for 18s | Applied Biosystems | Hs99999901_s1 | |
| MyiQ software | Bio-Rad Laboratories | ||
| Cleaved caspase-3 (Asp175) | Cell signaling technology | 9661 | |
| Anti rabbit IgG | Abcam | Ab6798 | |
| Sucrose | Sigma Aldrich | 84097 | |
| Vectashield | Vector Laboratories | ||
| Universal Microscope Axioplan 2 Imaging | Carl Zeiss | ||
| Paraformaldehyde | Sigma Aldrich | 441244 | |
| TBE buffer 10x | Promega | V4251 |
Referencias
- Brian, G., Taylor, H. R. Cataract blindness: challenge for the 21 st century. Bulletin of the World Health Organization. 79, 249-256 (2001).
- Jose, J. G., Pitts, D. G. Wavelength dependency of cataracts in albino mice following chronic exposure. Experimental Eye Research. 41, 545-563 (1985).
- Söderberg, P. G. Acute cataract in the rat after exposure to radiation in the 300 nm wavelength region. A study of the macro-, micro- and ultrastructure. Acta Ophthalmol. (Copenh). 66, 141-152 (1988).
- Meyer, L., Dong, X., Wegener, A., Söderberg, P. G. Dose dependent cataractogenesis and Maximum Tolerable Dose (MTD 2.3:16) for UVR – B induced cataract in C57BL/6J mice. Experimental Eye Research. 86, 282-289 (2008).
- McCarty, C., et al. Assessment of lifetime ocular exposure to UV-B: the Melbourne visual impairment project. Developments in Ophthalmology. 27, 9-13 (1997).
- Sasaki, H., et al. Localization of cortical cataract in subjects of diverse races and latitude. Invest. Ophthalmol. Vis. Sci. 44, 4210-4214 (2003).
- Söderberg, P. G. Experimental cataract induced by ultraviolet radiation. Acta Ophthalmol. (Copenh. 68, 1-77 (1990).
- Galichanin, K., Löfgren, S., Bergmanson, J., Söderberg, P. Evolution of damage in the lens after in vivo close to threshold exposure to UV-B radiation: cytomorphological study of apoptosis. Exp. Eye Res. 91, 369-377 (2010).
- McCarty, C. A., Taylor, H. R. A review of the epidemiologic evidence linking ultraviolet radiation and cataracts. Dev. Ophthalmol. 35, 21-31 (2002).
- Li, W. C., et al. Lens epithelial cell apoptosis appears to be a common cellular basis for non-congenital cataract development in humans and animals. Journal of Cell Biology. 130, 169-181 (1995).
- Michael, R., Vrensen, G., van Marle, J., Gan, L., Söderberg, P. G. Apoptosis in the rat lens after in vivo threshold dose ultraviolet irradiation. Investigative Ophthalmology and Visual Science. 13, 2681-2687 (1998).
- Ayala, M. N., Strid, H., Jacobsson, U., Söderberg, P. G. p53 Expression and Apoptosis In The Lens After Ultraviolet Radiation Exposure. Investigative ophthalmology of visual science. 48, 4187-4191 (2007).
- Galichanin, K., Wang, J., Lofgren, S., Soderberg, P. A new universal rat restrainer for ophthalmic research. Acta Ophthalmol. 89 (1), (2011).
- Ayala, M. N., Michael, R., Söderberg, P. G. In vivo cataract after repeated exposure to ultraviolet radiation. Experimental Eye Research. 70, 451-456 (2000).
- Söderberg, P. G., et al. Toxicity of ultraviolet radiation exposure to the lens expressed by maximum tolerable dose (MTD). Developments in Ophthalmology. 35, 70-75 (2002).
- Michael, R. Development and repair of cataract induced by ultraviolet radiation. Ophthalmic Research. 32, 1-45 (2000).
- Löfgren, S. . Cataract from ultraviolet radiation. , (2001).
- Ayala, M. N. . Influence of exposure patterns and oxidation in UVR induced Cataract. , (2005).
- Dong, X. . Safety limit estimation for cataract induced by ultraviolet radiation. , (2005).
- Löfgren, S., Michael, R., Söderberg, P. G. Impact of age and sex in ultraviolet radiation cataract in the rat. Investigative Ophthalmology and Visual Science. 44, 1629-1633 (2003).
- Ayala, M. N., Michael, R., Söderberg, P. G. Influence of exposure time for UV radiation-induced cataract. Investigative Ophthalmology and Visual Science. 41, 3539-3543 (2000).
Citar este artículo
Galichanin, K., Talebizadeh, N., Söderberg, P. Characterization of Molecular Mechanisms of In vivo UVR Induced Cataract. J. Vis. Exp. (69), e4016, doi:10.3791/4016 (2012).