Anders Fabian, Teister Julia, Funke Sebstian, Pfeiffer Norbert, Grus Franz, Solon Thanos, Prokosch Verena
Experimental Ophthalmology, Department of Ophthalmology, University Medical Center, Langenbeckstrasse 1, 55131, Mainz, Germany.
University Eye Hospital Mainz, School of Medicine, Langenbeckstrasse 1, 55131, Mainz, Germany.
Graefes Arch Clin Exp Ophthalmol. 2017 Jul;255(7):1395-1407. doi: 10.1007/s00417-017-3678-x. Epub 2017 May 24.
Clinical glaucoma is difficult to assess in terms of molecular pathophysiology, prompting studies in experimental models of glaucoma. The purpose of this study was to investigate quantitative changes in retinal protein expression at the onset of experimental glaucoma in rats. Analyzing the proteome provides a suitable tool to decipher the pathophysiological processes in glaucomatous degeneration.
Thermic cauterization of episcleral veins was utilized to elevate the intraocular pressure in Sprague Dawley rats. Morphological changes were surveyed on a cellular level with a staining of Brn3a-positive cells. The retinal nerve fiber layer was investigated using optical coherence tomography (OCT, Heidelberg Engineering) and the optic nerve was analyzed by an axonal grading system. Mass spectrometry-featured quantitative proteomics and immunohistochemical staining was used to identify specifically altered proteins in the course of intraocular pressure elevation and initial neurodegeneration. Proteomic data were further analyzed with Ingenuity Pathway Analysis and Cytoscape to analyze further molecular associations.
The intraocular pressure rose significantly (p < 0.001) for the follow-up period of 3 weeks after which animals were sacrificed. Eyes exposed to an elevated intraocular pressure showed an initial decrease of retinal ganglion cells, retinal nerve fiber layer (p < 0.05) and an impairment of the optic nerve (p < 0.01). Mass spectrometry led to the identification and quantification of 931 retinal proteins, whereas 32 were considerably altered. Bioinformatics-assisted clustering revealed that a majority of these proteins are functionally associated with cell differentiation, apoptosis and stress response. The creation of an interactive protein network showed that numerous altered proteins are connected regarding their cellular function. Protein kinase b, mitogen-activated protein kinase 1 and the NF-κB complex seem to be essential molecules in this context.
In conclusion, these results provide further lines of evidence that substantial molecular changes occur at the onset of the disease, identifying potential key players, which might be useful as biomarkers for diagnostics and development of medical treatment in the future.
临床青光眼在分子病理生理学方面难以评估，这促使人们对青光眼实验模型展开研究。本研究的目的是调查大鼠实验性青光眼发病时视网膜蛋白表达的定量变化。分析蛋白质组为解读青光眼性退变的病理生理过程提供了一个合适的工具。
采用巩膜静脉热烧灼法升高斯普拉格-道利大鼠的眼压。通过对Brn3a阳性细胞进行染色，在细胞水平上观察形态学变化。使用光学相干断层扫描（OCT，海德堡工程公司）研究视网膜神经纤维层，并通过轴突分级系统分析视神经。采用以质谱为特色的定量蛋白质组学和免疫组织化学染色来鉴定眼压升高和初始神经退变过程中特异性改变的蛋白质。利用Ingenuity通路分析和Cytoscape软件进一步分析蛋白质组学数据，以分析更多的分子关联。
在为期3周的随访期内眼压显著升高（p < 0.001），之后处死动物。眼压升高的眼睛显示视网膜神经节细胞、视网膜神经纤维层最初减少（p < 0.05），视神经受损（p < 0.01）。质谱分析鉴定并定量了931种视网膜蛋白，其中32种有显著变化。生物信息学辅助聚类显示，这些蛋白质中的大多数在功能上与细胞分化、凋亡和应激反应相关。创建的交互式蛋白质网络表明，许多改变的蛋白质在细胞功能方面相互关联。在这种情况下，蛋白激酶b、丝裂原活化蛋白激酶1和NF-κB复合物似乎是关键分子。
总之，这些结果提供了更多证据，表明在疾病发病时会发生大量分子变化，确定了潜在的关键分子，这些分子未来可能作为诊断和治疗药物开发的生物标志物。
