Background: Glaucoma is a progressive optic neuropathy characterized by degeneration of

Background: Glaucoma is a progressive optic neuropathy characterized by degeneration of neurons due to loss of retinal ganglion cells (RGCs). expression of 1-integrin, phosphorylation of focal adhesion kinase (FAK) and protein kinase B (PKB, or AKT) were investigated with real-time polymerase chain reaction and Western blotting analysis. Results: Elevated hydrostatic pressure induced apoptosis in cultured NVP-AEW541 RGC-5 cells. Pressure with 40 mmHg for 24 h induced a maximum apoptosis. Laminin was declined in RGC-5 cells after exposing to 40 mmHg for 24 h. After pretreating with laminin, RGC-5 cells survived from elevated pressure. Furthermore, 1-integrin and phosphorylation of FAK and AKT were increased compared to 40 mmHg group. Conclusions: The data show apoptosis tendency of RGC-5 cells with elevated hydrostatic pressure. Laminin can protect RGC-5 cells against high pressure via 1-integrin/FAK/AKT signaling pathway. These results suggest that the decreased laminin of RGC-5 cells might be responsible for apoptosis induced by elevated hydrostatic pressure, and laminin or activating 1-integrin/FAK/AKT pathway might be potential treatments to prevent RGC loss in glaucomatous optic neuropathy. studies revealed the characteristic apoptosis of RGCs in response to elevated intraocular pressure (IOP)[7,8,9] and NVP-AEW541 there has been some research on the direct effect of IOP on RGC apoptosis. Agar < 0.05 was considered to represent statistical significance. All statistical analyses were performed with SPSS version 20.0 software (SPSS Inc., Chicago, IL, USA). RESULTS Elevated hydrostatic pressure induces retinal ganglion cell apoptosis Flow cytometry analysis was used to detect RGC-5 cell survival at different time with 0, 20, 40, and 60 mmHg [Figure 1a]. The histogram indicates that a pressure of 40 mmHg for 24 h induced maximum apoptosis, 7.63% 1.02% in early apoptosis and 17.93% 1.66% in advanced mortality apoptosis (< 0.001 vs. 0, 20, and 60 mmHg group for 24 h; < 0.001 vs. 40 mmHg group at 6 and 12 h) [Figure 1b]. MTT cell proliferation assay was performed to assess the activity of RGC-5 cells treated with hydrostatic pressure. Cell viability was as follows: 88.67% 5.40% (20 mmHg), 52.70% 2.00% (40 mmHg), and 80.62% 7.91% (60 mmHg). Apparently, the MTT results showed that the cell survival rate decreased at 40 mmHg for 24 h (= 0.002 vs. 20 mmHg, and = 0.010 vs. 60 mmHg) [Figure 1c]. Moreover, we analyzed the expression of cleaved caspase-3, which was significantly increased with 40 mmHg for 24 h (< 0.001 vs. 0, 20, and 60 mmHg) [Figure ?[Figure1d1d and ?and1e].1e]. These results suggested that there was a much greater injury to RGC-5 cells with 40 mmHg for 24 h than that was seen in other experimental groups. Figure 1 Hydrostatic pressure regulated survival of retinal ganglion cell-5 cells. (a) Flow cytometry of cell IL6 apoptosis probed by Annexin V binding (horizontal: FITC-A) and propidium iodide exclusion (vertical: Propidium iodide-A). (b) Percentages of apoptotic NVP-AEW541 … Laminin expression and function in cells exposed to 40 mmHg pressure In cultured RGC-5 cells, NVP-AEW541 laminin was localized to the cell membranes using immunofluorescence (IF) [Figure 2a]. Moreover, IF staining revealed a marked change of laminin in the pressure group compared with the normal group. The fluorescent intensity of laminin was less prominent in the 40 mmHg for 24 h group compared to the control group (= 0.010) [Figure 2b]. To determine whether laminin plays a protective role in RGC-5 cells under elevated hydrostatic pressure, cells incubated and pretreated with 10 g/ml laminin were exposed to 40 mmHg for 24 h. Apoptosis was detected by Annexin-V and PI staining and Western blotting of cleaved caspase-3 proteins. Surprisingly, flow cytometry analysis showed that survival was arrested in the.

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