Objective To observe the relationship between retinal microglial activations and ganglion cell (RGC) damages in early-stage diabetic rats. Methods A total of 20 SpragueDawley（SD）rats were randomly divided into 4 groups (each with 5 rats): 1 month control group, 1 month diabetes group, 3 month control group, 3 month diabetes group. Diabetes was induced by intraperitoneal injection of streptozotocin (STZ). The RGCs of all rats were retrograde labeled by carbocyanine dye DiI injected at the superior colliculi.Microglial cells and RGCs in retinal flat-mounts and sections were stained immunohistochemically and recorded under confocal microscope.Results The diabetic microglial cells were amoeboid and ovoid with fewer processes on retinal flat mounts. The density of microglial cells which phagocytosed DiI particles in the RGC layer significantly increased in the 3month diabetes group（P＜0.01）. The density of microglial cells in the RGC layer significantly increased in the 1- and 3- month diabetes group（P＜0.05）. However there were more microglial cells in the RGC layer in the 3- month diabetes group than the 1-month diabetes group（P＜0.0001）. Significant correlation was found between the amount of microglial cells and that of RGCs in the early-stage of diabetes. Conclusions Microglial cell activation has close relationship with the RGC damages in early-stage diabetic rats.
ObjectiveTo observe the role of Notch signaling pathway inhibitor in differentiation process of stem cells derived from retinal Müller cells into the ganglion cell. MethodsRetinas of Sprague Dawley rat at postnatal 10-20 days were dissociated from eye balls. The third passage of Müller cells was used in this experiment, which cultured by repeated incomplete pancreatic enzyme digestion method. The retinal Müller cells were induced in the serum-free dedifferentiation medium. The cell proliferation state was observed under an inverted microscope. The expression of the specific markers Nestin and Ki-67 of retinal stem cells was measured by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. The positive rate of nucleus was detected by Edu. The retinal stem cells was divided into Gamma secretase inhibtor-I (GSI) group and control group, the rate of ganglion cells was counted by using immunofluorescence staining. ResultsThe cell proliferation had gathered to form a sphere. Immunofluorescence staining showed that the expressions of Nestin and Ki-67 were (92.94±6.48%) and (85.96±6.04%) respectively. Edu positive rate of nucleus was (82.80±6.65)%. RT-PCR and Western blot further confirmed the high expression of Nestin and Ki-67 in the cell spheres but not in the Müller cells. The positive rate of ganglion cells were (16.98±2.87)% and (11.17±0.71)% in GSI group and control group respectively, with the significant difference (t=3.210, P=0.002). ConclusionNotch signaling pathway is an important regulatory gene in stem cells differentiated into retinal ganglion cell.
ObjectiveTo investigate the effects of FTY720 on retinal photoreceptor cells and microglial following light-induced degeneration in rat retina. Methods120 Sprague-Dawley rats were randomly divided into four groups including FTY720 group, solvent control group, model group and normal group. The rats of normal group were not intervened. The FTY720 group, solvent control group and model group establish retinal light injury mode. FTY720 was injected into abdominal cavity of the rats in FTY720 group 0.5 hours before light exposure. 50% dimethylsulfoxide was injected into abdominal cavity of the rats in solvent control group. The expressions of microglial cells in rat retinal were quantified using flow cytometry, the expressions of interleukin (IL)-1βwere examined by enzyme-linked immuno sorbent assay at 6 hours, 1 day, 3 days, 7 days after light exposure. The apoptosis of retinal photoreceptor cells were measured by terminal-deoxynucleoitidyl transferase mediated nick end labeling at 1 day after light exposure. The morphological change of retinal were viewed by haematoxylin and eosin staining at 7 days after light exposure. ResultsThe expressions of microgilal and IL-1βbegan to rise at 1 day after light exposure, reached at peak at 3 days and decreased at 7 days. The expressions of IL-1βand microglial in FTY720 group were significantly lower than solvent control group and model group, but higher than normal group (P < 0.05).One day after exposure to light, the apoptosis cell ratio in normal group, model group, solvent control group and FTY720 group were 0, (87.66±2.50)%, (86.00±2.44)%, (49.66±2.80)%. The apoptosis cell in FTY720 group were higher than normal group, lower than solvent control group and model group (P < 0.05). Seven days after exposure to light, the retinal in normal group was structured and the cell was arranged well, the cell in solvent control group and model group was irregular arrangement and the outer nuclear layer (ONL) was thin after light exposure. The thickness of the ONL in FTY720 group was significantly higher than solvent control group and model group, below normal group. ConclusionFTY720 can prevents retinal photoreceptor cells from apoptosis and inhibits activation of microglial.
Objective To investigate the cellular viability and mitochondrial reactive oxygen species (ROS) production of the Müller cells under high glucose condition, and explore the protection role of the 5,6-dihydrocyclopenta-1, 2-dithiole-3-thione (CPDT) on Müller cells. Methods Müller cells from Sprague Dawley rats were divided into 5 groups randomly, including 25 mmol/L normal glucose group (group A) and 65 mmol/L high glucose group (group B). High glucose group with 45, 60, 70 μmol/L CPDT and cultured them 72 hour was set as group C, D and E. Water soluble tetrazolium salt (WST)-8 was used to measure the cellular viability. Flow cytometry was used to measure the active oxygen and apoptosis index. The expression of nuclear factor erythroid 2-related factor 2 (Nrf2), hemeoxygenase-1 (HO-1), Bcl-2 and Bax protein were measured by Western blot. Results Compared with group A, the WST-8 showed that the viability of Müller cells apparently decreased in group B (t=39.59,P＜0.05). Compared with the group B, the viability of Müller cells had changes in group C (t=0.97,P＞0.05), but recovered in group D and E (t=−4.17, −7.52;P＜0.05). Compared with group A, the FCM showed that the mitochondrial ROS levels was higher in group B (t=−30.99,P＜0.05). Compared with group B, the mitochondrial ROS levels were decreased in group D (t=27.68,P＜0.05). Compared with group A, Bax, Nrf2 and HO-1 increased (t=–11.03, –63.17, –11.44;P＜0.05), while the bcl-2 decreased in group B (t=7.861,P＜0.05). Compared with the group B, Nrf2, HO-1 and Bax decreased (t=15.11, 26.59, 6.27;P＜0.05), while the bcl-2 increased in group D (t=−6.53,P＜0.05). Conclusions Under the high glucose, CPDT may reduce the mitochondrial ROS levels and the expression of Nrf2, HO-1 and Bax protein of Müller cells. It may inhibit apoptosis through activating the Nrf2/HO-1 pathway and balancing of level of Bcl-2 protein and mitochondrial ROS.
Retinal macrophages and (or) microglial cells play important roles in regulating inflammation, angiogenesis and tissue repairing, thus affect the development and prognosis of ischemic retinal disease, ocular immune diseases and ocular tumors. Reversing the polarization imbalance of these cells may provide new therapeutic strategies for ischemic retinal disease and ocular immune diseases. The duality of the polarization direction of these cells is still controversial in the inflammatory reaction and pathological angiogenesis of ischemic retinal disease. Meanwhile, the plasticity and diversity of the function need to be further studied and discussed.
Objective To observe the effect of netrin-1 on retinal Müller cells in diabetes mellitus (DM) rats. Methods Fifty Sprague-Dawley rats were randomly divided into the normal control group (group A), normal + balanced salt solution (BSS) group (group B), normal+netrin-1 group (group C), DM+BSS group (group D) and DM+netrin-1 group (group E), with 10 rats in each group. DM rats were induced by intraperitoneal injection of Streptozotocin (60 mg/kg). The expression level of glial fibrillary acidic protein (GFAP) on retinal Müller cells was determined by immunohistochemistry, the level of GFAP mRNA was analyzed by real-time fluorescence quantitative reverse transcription polymerase chain reaction. Results Immunohistochemistry showed that GFAP was distributed in retinal ganglion cells and retinal nerve fiber layer in group A, B and C. Compared to group B, GFAP staining was brighter in the group D. There were significant differences in the expression of GFAP protein and mRNA among groups A-E (F=203.43, 72.91; P=0.00, 0.00), they were higher in group D than group A (t=−26.01, 22.26; P=0.00, 0.00), and group E (t=−10.78, 3.93; P=0.00, 0.00). They were higher in group E than group A (t=7.00, −9.82; P=0.00, 0.00). There were no significant differences in between group A and group C (t=−0.29, 0.50; P=0.77, 0.62). Conclusion The expression of GFAP in Müller cells of DM rats could be decreased by injecting netrin-1 into vitreous.