| Objective: Rabbits with chronic high intraocular pressure were establishhed in this study to investigate the effect of chronic high intraocular pressure on profile of retinal protein expression by two-dimensional electrophoresis (2-DE), and Liquid Chromatographic Chip Mass Spectrometry (LC-CHIP-MS/MS) to throw new light on mechanism research that how glaucoma injure to retinal ganglion cells (RGCs) and treatment.Methods: 1. Animal model: 0.2 ml carbomer solution injection to the left anterior chamber of experimental animals to produce chronic high intraocular pressure model, whereas the right eye for the normal control. If the models keep intraocular pressure > 22 mmHg for one week, we take that for granted. If intraocular pressure <22 mmHg, procedure was repeated as above after 7 days. After the modeling, intraocular pressure was measured 2 times per week. Eyes of the model animals were removed after28 days of the construction, and protein of retinal tissue was extracted from retinal tissue.2. 2-DE: Performed as guideline of Bio-Rad two-dimensional electrophoresis test. The results of 2-DE separation were performed image analysis after the classic silver staining compatible with MS, to detect difference by PDQest7.4.0 image analysis software. Two samples with the same protein that with more than 2 times of the optical density differences was considered as different of the expression.3. By LC-CHIP-MS/MS analysis, mass spectrograms were developed. Differentially expressed proteins were searched in the UniProtKB/ SWISS-PROT and IPI-International Protein Index database using the Spectrum Mill to identify and access their biological information.Results: 1. In the group of Chronic high intraocular pressure. intraocular pressure(IOP) began to increase after 3 h of construction, and reach 40 mmHg or more after about 14 d . Then most of the IOP remained at 30 ~ 40 mmHg. In normal control group, IOP maintain normal at an average of 13 ~ l5 mmHg throughout the experiment.2. There are 9 proteins differentially expression, 3 of which were significant by up-regulated. LC-CHIP-MS/MS identified three proteins as: heat shock protein 70 (heat shock 70 kD protein, HSP70), pyruvate kinase and enolase.Conclusion: 1. Carbomer solution injection to anterior chamber is an ideal means to construct animal models with chronic high intraocular pressure.2. 2-DE and LC-CHIP-MS/MS analysis showed that compared with normal control eyes, the quality and quantity of expression of retinal proteins in chronic high intraocular pressure group changes which involve several groups of proteins related to RGCs glycolysis and stress response. Our findings indicate that these proteins may be involved in the process of RGCs apoptosis in chronic glaucoma. |
