| BACKGROUDIn ischemic retinal diseases, VEGF, which is synthesized and secreted by all kinds of retinal cells, not only promotes vasculogenesis, increases permeability of the blood retinal barrier, but also influences all retinal cells. Although VEGF and its inhibitors are applied for clinical treatments, their detail mechanisms on retinal cells have been unclear thus far, e.g. how they effect on Müller cells. Müller cell, as a principal glial cell of the retina, plays an important role in maintenance of the retinal structure, metabolism and function. Reactive Müller cell gliosis in ischemic retinal diseases is regulated by various cellular factors (e.g., VEGF) from surrouding retinal cells. In present studies, we observed effects of VEGF on rat Müller cells under hypoxia in vitro.AIM1. To establish a hypoxia model of Müller cells using CoCl2, and to observe the effects of hypoxia on rat Müller cells in vitro.2. To observe the effects of VEGF on rat Müller cells under hypoxia in vitro.METHODS1. Müller cell culture and identification: Newborn rat retinas within 1 wk were minced and digested in the typsin solution, the suspension was fitered through a 53μm nylon mesh. After the filtrate was collected and centrifuged, mixed cells were resuspended and seeded onto culture flasks. The cells were purified by a modified digestion method for 2～3 times. The Müller cells of passage 2～3 were characterized for the expressions and localizations of GFAP and glutamate synthase with immunofluorescence staining.2. Establishement of a hypoxia model of Müller cells using CoCl2: The cells of passage 3～5 purified and identified were exposed to 200 mM of CoCl2 for 0, 4, 8, 12, 16, 20 and 24 h. The Müller cells under normoxia were used as controls. The effect of hypoxia on the proliferation of cultured Müller cells was determined by MTT assay. The effects of hypoxia on the expressions of GFAP, VEGF, Flt-1 and Flk-1 of Müller cells were also examined with immunohistochemistry staining and integrated absorbence (IA) assay. The cell apoptosis levels were detected by TUNEL method.3. Effects of VEGF with various concentrations on Müller cells under hypoxia: The rat Müller cells were cultured for 24 h in the medium with 10～100μg/L of VEGF. The cells were then exposed to 200 mM of CoCl2 for 24 h. MTT method was used to evaluate their viability and cell apoptosis was detected by TUNEL method.4. Effects of 75μg/L of VEGF on Müller cells after various hypoxia periods: Müller cells were cultured for 24 h in the medium with 75μg/L of VEGF. These cells were exposed to 200 mM CoCl2 for 0, 4, 8, 12, 16, 20 and 24 h, and then tested with MTT assay and TUNEL staining.5. Effects of SU1498 on Müller cells under hypoxia: Müller cells were cultured for 24 h in the medium with 75μg/L of VEGF and 700μg/L of SU1498. These cells were exposed to 200 mM CoCl2 for 24 h, and then tested by MTT assay and TUNEL staining.RESULTS1. The rat Müller cells were purified by digestion of the trypsin solution. The cells showed positive fluorescence staining for GFAP and glutamate synthase. The purity of passage 3 was 90%, and its viability was 87.3%.2. Compared with the normoxia control group, hypoxia increased the expression of GFAP in Müller cells (P<0.05). IA levels at 0, 4, 8, 12, 16, 20 and 24 h of hypoxia were 1933±79, 2005±98, 2451±87, 2396±86, 2092±129, 1892±63 and 1659±55, respectively. The cells showed swelling and suspended in the culture medium at 12 h of hypoxia, but the cell viability was still 83% at 16 h under hypoxia. The VEGF protein level was upregulated significantly under the treatment of hypoxia (P<0.01) and reached 48.4±1.4 at 24 h. Flk-1 and Flt-1 levels were 2.3±0.5 and 2.0±0.7 in normal conditions respectively. The Flk-1 level increased to 2.3±0.5, 4.1±0.2, 5.1±2.3, 10.3±3.1, 12.5±3.3, 17.2±3.2 and 18.4±3.4 and Flt-1 level increased to 2.0±0.7, 4.2±1.5, 17.4±1.5, 25.4±2.3, 30.2±1.8, 31.6±2.2 and 33.5±1.9 at 4, 8, 12, 16, 20 and 24 h under hypoxia, respectively. At 24 h under hypoxia, the cell viability decreased to 72.7% and the apoptosis index increased 8.2 fold compared with those of normoxia control group.3. Müller cells were cultured in medium with 10～100μg/L of VEGF before hypoxia. The cell viability increased and the apoptosis index decreased dose-dependently after the treatment with 50～100μg/L of VEGF (P<0.05). After the treatment with 75μg/L and 100μg/L of VEGF, the cell viability levels were 0.201±0.031 and 0.199±0.007, and the apoptosis index was 12±1.2 and 11±1.5, respectively. There were significanct differences between the parameters of treated groups and those of the control group(P<0.05), but the parameters among these treated groups were not different significantly (P>0.05). The cells were treated with 75μg/L of VEGF under hypoxia for 0～24 h. We found that the cell viability increased and the apoptosis index reduced. The cell viability increased 1.3 fold and the apoptosis index reduced to two third at 24 h after hypoxia, compared with the control group (P<0.05).4. Exposed to hypoxia, the cell viability was 0.168±0.005 and 0.129±0.002, and the apoptosis index was 14±1.8 and 12±1.5 after the treatment with VEGF+SU1498 and VEGF, respectively. SU1498 also showed a significant inhibitory effect on the Müller cells compared with the cells under hypoxia without treatment of VEGF and SU1498 (P<0.05).CONCLUSION1. Temporary hypoxia treatment might stimulate the proliferation of the Müller cells and increase cell viability. The persistence of hypoxia might result in cell damage. Expressions of GFAP, VEGF and its receptors were enhanced after hypoxia treatment.2. VEGF could protect Müller cells from hypoxia and Flk-1 might be a key element in molecular pathway of the protection.The innovation of this experiment: The study oberserved firstly the effects of VEGF on the rat Müller cells under hypoxia in vitro. Similar reseach has not yet been in the literature.
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