The Effect Of Minocycline On Pressure-treated Retinal Neurons In Vitro

With the increase of aging population, the promotion of eye-sight screening and the increase of popular concern, more cases of glaucoma have been observed and the number is still going up, which globally makes this disease the major blinding disease of adults. Glaucoma is characterized by the progressive death of retinal neurons (RNs), and the pressure-dependent neurons-damage is the main cause of this disease. Therefore reducing the intraocular pressure has become the primary concern, and the treatments alleviating and sparing neurons-damage have also gained more and more attentions nowadays. Generally spoken, neuroprotection refers to all treatments that could protect RNs from apoptosis. According to reported research, this kind of optical neuroprotection could be achieved through drugs application. Minocycline (MINO) is a member of the tetracycline class of molecules with broad-spectrum antibiotic activity, and it has been confirmed to be capable of neuroprotection in a number of models of neurodegeneration disease.In present study, the model of pressure-treated rat RNs in vitro is successfully constructed. Through cellular morphology observation, MTT colorimetric method testing cell vitality, the neuroprotection capability of MINO is evaluated; and through AO/EB double staining method testing cell apoptosis rate, then through the comparison of the change of iNOS and caspase-3 expression with immuno-cellular-chemistry methods, the mechanism of MINO's protection on pressure-treated rat RNs could be analyzed. And the possible mechanism of this nerve-protection could also be initially explained. This research is divided into three parts. PartⅠConstruction of pressure-treated retinal neurons (RNs) culture model in vitro[Objective] To develop a method for constructing pressure-treated retinal neurons (RNs) culture model for further experiments on glaucoma.[Methods] With trypsin digestive method, rat retinals (newborn 1-3 days) were acquired and then made into cell suspension, and inoculated into culture flasks (with cover glasses previously coated with polylysine), then cultured in incubator at 37℃and 5% CO2. Non-neuron cells growth were inhibited. On 3rd day of cell culture, a portion of cultured cells were taken for immuno-cellular chemistry exam with anti-rat MAP2 multiclone antibody and anti-rat THY1.1 monoclone antibody, in order to identify RNs and retinal ganglion cells (RGCs). 2 groups were set for this part of research: (A) the control group ; (B) the pressure-treated group, the pressure-treated group were cultured under 60mmHg for 24 hours. The cell morphology was observed with inverted phase contrast microscope, and the cell vitality was analyzed with MTT method.[Results] The growth of RNs in vitro was satisfying, a certain amount of the cells developed neurites and some tangled into net-shape, the purity of RGCs was 53.33%. An obvious change in the morphology of pressure-treated group has been observed, and MTT staining method showed that the vitality of cells has significantly decreased (P<0.05).[Conclusion] The model of pressure-treated RNs culture in vitro has been successfully constructed, and there was significant pressure-induced damage in pressure-treated group.PartⅡThe effect of multi-level concentrations of minocycline on livability of pressure-treated RNs[Objective] To evaluate the protective effects of different concentrations of MINO on RNs cultured in vitro under pressure.[Methods] Rat RNs cultured in vitro were divided into the control group, the pressure-treated group and the MINO treated groups (5 different concentrations of MINO in each group, as 0.002, 0.02, 0.2, 2, 20μmol/L). By comparing the cell morphology and MTT results, the protective effects of different concentrations MINO on these cells were evaluated.[Results] Compared with pressure treated group, the MINO treated groups got higher cell vitality, and the high level concentration group was better than low level group, which indicated that certain concentration of MINO could significantly alleviate the pressure-induced cell damage.[Conclusion] MINO could improve the survival rate of RNs cultured in vitro under pressure, and performed an obvious protective effect.PartⅢThe mechanism of minocycline's protection on pressure-damaged RNs[Objective] To analyze if the mechanisms of MINO's protection on pressure-damaged RNs involve apoptosis.[Methods] Rat RNs were cultured and then divided into the control group, the pressure-treated group and the MINO treated groups (5 different concentrations of MINO in each group, 0.002, 0.02, 0.2, 2, 20μmol/L). The apoptosis rate of these cells was observed with AO/EB double staining method, then the expression of intra-cellular caspase-3 and iNOS was measured.[Results] A large amount of RNs suffered from apoptosis in pressure-treated group, and this situation in MINO treated groups were reversed. This effect was confirmed as dose-dependent, while 0.2–20μmol/L of MINO was the most effective. Intra-cellular caspase-3 and iNOS expression were remarkably high in the pressure-treated group, and 20μmol/L of MINO could significantly reduce both expressions.[Conclusion] MINO could effectively inhibit the pressure-induced RNs apoptosis, and this effect must be one of the mechanisms of exerting the neuroprotection.