Protective effects of ciliary neurotrophic factor on the retinal ganglion cells by injure of hydrogen peroxide.

AIM: To explore the effect of ciliary neurotrophic factor (CNTF) on retinal ganglion cell (RGC)-5 induced by hydrogen peroxide (H2O2). METHODS: After cell adherence, RGC-5 culture medium was changed to contain different concentrations of H2O2 from 50 to 150 µmol/L at four time points (0.5, 1, 1.5 and 2h) to select the concentration and time point for H2O2 induced model. Two different ways of interventions for injured RGC-5 cells respectively were CNTF as an addition in the culture medium or recombinant lentiviral plasmid carrying CNTF gene transfecting bone mesenchymal stem cells (BMSCs) for co-culture with RGC-5. RESULTS: Compared to the control group, H2O2 led to RGC-5 death closely associated with concentrations and action time of H2O2 and we chose 125 µmol/L and 2h to establish the H2O2-induced model. While CNTF inhibited the loss of RGC-5 cells obviously with a dose-dependent survival rate. Nevertheless two administration routes had different survival rate yet higher rate in recombinant lentiviral plasmid group but there were no statistically significant differences. CONCLUSION: Both the two administration routes of CNTF have effects on RGC-5 cells induced by H2O2. If their own advantages were combined, there may be a better administration route.

In vitro induction and differentiation of umbilical cord mesenchymal stem cells into neuron-like cells by all-trans retinoic acid.

AIM: To determine the optimal concentration for inducing the differentiation of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) into neuron-like cells, although it is understood that all-trans retinoic acid (ATRA) regulates cell proliferation in the nervous system by modulating the balance between mitosis and apoptosis. METHODS: The abilities of ATRA to promote apoptosis as well as neural differentiation were assessed in cultured hUC-MSCs by morphological observation, MTT assay, annexin V-FITC/PI flow cytometry and immunocytochemistry. RESULTS: The data showed that low concentrations of ATRA (0.5 µmol, 0.25 µmol) had no effect on the number of cells. However, treatment with 1.0 µmol or 2.0 µmol ATRA induced a 24.16% and 52.67% reduction in cell number, respectively, compared with vehicle-treated cultures. Further, 4.0 µmol ATRA had a potent effect on cell number, with almost no adherent cells recovered after 24h. We further showed that 0.5 µmol ATRA caused these cells to express characteristic markers of neuronal progenitor cells. CONCLUSION: Taken together, we conclude that ATRA has a dose-dependent influence on the neural differentiation and apoptosis of hUC-MSCs. These findings have implications on the use of ATRA-differentiated hUC-MSCs for the study of neural degeneration diseases.

[Differentiation and apoptosis effects of all-trans retinoic acid on inducing umbilical cord mesenchymal stem cells into neuron-like cells].

OBJECTIVE: To evaluate the role of different concentration of all-trans retinoic acid (ATRA) on the morphology, proliferation and apoptosis in inducing umbilical cord mesenchymal stem cells (MSC) into neuron-like cells in vitro, and screen the optimal concentration of ATRA. METHODS: It was an experimental study. The third passage of MSC was placed in 24-well cell culture plates at a density of 1 × 10(4)/well. After the adherent of cells, the medium was changed to DMEM/F-12 containing different concentration of ATRA (0.25 µmol/L, 0.5 µmol/L, 1.0 µmol/L, 2.0 µmol/L, 4.0 µmol/L) for 24 h respectively. The cells cultured without ATRA were taken as the control group. After another 24 h, the morphologic changes of induced cells were observed by inverted microscope and cell proliferation, apoptosis of ATRA was analyzed using the MTT colorimetric assay. We take another control group and ATRA groups to detect the apoptotic and positive stained percentage of induced cells by Annexin V-FITC/PI combining flow cytometry. The optimal concentration of ATRA was determined by all the above-mentioned index. According to the nature of the material, analysis of variance (ANOVA) was employed for absorption value and apoptosis rate in different concentration of ATRA for 24 h, t test for further comparison between two groups. T-test were also used between the positive expression of induced neuron-like cells and the control group. RESULTS: Compared to the control group, ATRA at the concentration of 0.25 µmol/L did not inhibit the proliferation of umbilical cord MSC obviously (t = 0.72, 1.32, P > 0.05). Part of MSC were floating instantly at the moment of adding ATRA of 4.0 µmol/L and no adherent cells were observed after 24 h' culture. Exposed to ATRA at the concentration of ≥ 1.0 µmol/L for 24 h, the proliferation of MSC were significantly inhibited, showing a dose-dependent manner (t = 8.8, 18.9, 22.1; P < 0.01). 0.5 µmol/L of ATRA did not affect the proliferation of cells and its morphology remained normal; 1.0 µmol/L of ATRA affected very few cells; but 2.0 µmol/L of ATRA cultured for 24 h inhibited the proliferation of cells obviously than 1 h, and the cells increased in size and became flattened. Flow cytometry showed that the rate of apoptosis between the control group and ≥ 1.0 µmol/L groups were significantly different (t = 9.88, 19.95, 31.61; P < 0.01). CONCLUSION: In the process of inducing umbilical cord MSC into neuron-like cells, 0.5 µmol/L ATRA was the optical concentration. ≥ 1.0 µmol/L ATRA can inhibit the cell proliferation, increase the apoptosis of cells significantly and caused obvious damages.

Epidermal growth factor promotes the differentiation of stem cells derived from human umbilical cord blood into neuron-like cells via taurine induction in vitro.

Results of recent investigations have demonstrated the plasticity of mesenchymal stem cells (MSC) can differentiate into neural lineages. In this study, we explored the experimental condition of differentiation into neuron-like cells or rhodopsin (RHOS)-positive cells induced by epidermal growth factor (EGF) and taurine in vitro and to investigate their biological characteristics. MSC were obtained from umbilical cord blood (UCB) of term deliveries. Cultured cells were treated with Dulbecco's modified Eagle's medium/F12 (pH 7.0-7.2) supplemented with 30 ng/ml EGF. After the third cell passage, the cells were trysinized and analyzed with a flow cytometer using the following monocloned antibodies: CD90, CD29, CD34, CD44, and CD45. Taking another MSC of the third passage, its basal medium was replaced with alpha minimum essential medium supplemented with taurine (50 micromol/L). Cells were cultured for an additional 8-10 d, fixed, and then immunocytochemically analyzed. Primary antibodies included the following: neuron-specific enolase (NSE), RHOS, and nestin. In our study, we isolated a cell population derived from UCB, which possesses morphological characteristics similar to those of MSC isolated from bone marrow. In the cytometric analysis, MSC did not present labeling for the hematopoietic line (CD34 and CD45) and were positive for CD29, CD44, and CD90. After induction by taurine, 80.5 +/- 16.2% of the cell population expressed NSE, 36.8 +/- 9.6% expressed RHOS, and 29.6 +/- 9.3% expressed Nestin, while only 7.9 +/- 3.5% expressed NSE in the control group. This study demonstrates that partial MSC induced by taurine and EGF can differentiate into neuron-like cells or RHOS-positive cells in vitro, which may provide a promising therapeutic strategy for the treatment of some forms of retinal degeneration.

Changes in visual acuity, axial length, and refraction after removal of intraocular silicone oil following retinal reattachment surgery in Chinese patients: An open-label, prospective study.

BACKGROUND: As a vitreous substitute for long-term tamponade, silicone oil is widely used in vitreoretinal surgery to treat retinal detachment with proliferative vitreoretinopathy and some internal reconstruction after globe trauma. OBJECTIVES: The aim of this study was to compare the changes in visual acuity, axial length, and refraction in eyes before and after removal of intraocular silicone oil of 2 different viscosities after retinal reattachment surgery. The difference in the final anatomic success (stable retinal reattachment) rate was also assessed. METHODS: Patients with surgically reattached retinas were enrolled in this open-label, prospective, nonrandomized study. All patients underwent pars plana vitrecto-my, lensectomy, scleral buckling or encircling, and epiretinal membrane dissection; silicone oil was removed after stable retinal reattachment was achieved. Refraction, axial length, final visual acuity, and stable retinal reattachment were assessed ≤2 days prior to surgery (baseline) and ≤1 month after silicone oil removal. Refraction was measured using an autorefractometer, and axial length was measured using A-scan ultrasonography, while visual acuity was assessed using a standard Snellen chart. RESULTS: Of the 96 eyes assessed for inclusion, 89 eyes of 89 Chinese patients (mean [SD] age, 36.8 [4.3] years) were included in the study. Forty-two eyes (47.2%) were filled with 3700-centistoke (cS) silicone oil and 47 (52.8%) were filled with 5000-cS silicone oil. The mean interval between instillation and removal of the silicone oil was similar between the 3700-cS and 5000-cS groups (5.37 vs 5.10 months, respectively). The mean changes in visual acuity from before surgery to after removal of the silicone oil in the 3700-cS and 5000-cS groups were not significantly different (13/100 vs 15/100). The mean increase in axial length was also not significantly different in the 3700-cS group compared with the 5000-cS group (11.92 [1.97] vs 12.33 [1.28] mm). Mean decrease in refraction was significantly lower in the 3700-cS group compared with the 5000-cS group (5.80 [1.51] vs 6.88 [2.31] diopters; t = 2.57, P < 0.05). The anatomic success rate was 92.9% (39/42 patients) in the 3700-cS group and 91.5% (43/47) in the 5000-cS group. CONCLUSIONS: A statistically significant decrease in refraction from baseline was found in the 3700-cS group compared with the 5000-cS group in these Chinese patients who underwent instillation and removal of silicone oil after retinal reattachment surgery. There were no other statistically significant differences between the 2 groups.