Objective To observe the permeability and stability of the transfection of antisense oligonucleotide (ASODN) hybridized epidermal growth factor receptor (EGFR) to retinal glial cells (RG).Methods Phosphorothioate and unmodified EGFR ASODN conjugated with 5′-isothioc yanate (5′-FITC) were encapsulated with or without lipofectin, and then added into human retinal glial cells culture media. The cellular permeability and stability of the transfection were observed by fluorescence microscopy in fixed cells.Results In the absence of lipofectin, phosphorothioate and unmodified EGFR ASODN were found in a few RG cells at 30 minutes, and in about 50% RG cells at 4 hours. Phosphorothioate EGFR ASODN were kept in RG cells for 3-4 hours and disappeared at about 8 hours. In the presence of lipofectin, phosphoro thioate and unmodified EGFR ASODN were found in a few RG cells at 15 minutes and about 70%-80% RG cells at 4 hours. Phosphorothioate EGFR ASODN were kept in cells for 10-12 hours, and phosphorothioate and unmodified EGFR ASODN were disapp eared at about 14 hours and 4 hours respectively.Conclusion 5′-FITC EGFR ASODN encapsulated with lipofectin could enter RG cells and express stably in RG cells. (Chin J Ocul Fundus Dis，2003，19：52-54)
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.
Objective To observe the expression of related proteins of retina after subretinal implantation with inactive chips.Methods A total of 27 healthy adult New Zealand white rabbits were randomly divided into three groups: operation group (12 rabbits) in which the rabbits were implanted with inactive chips into the interspace beneath retina;shamoperation group (12 rabbits) in which the rabbits were implanted with inactive chips into the interspace beneath retina which was taken out immediately;the control group (3 rabbits). Animals were sacrified for immunohistological study 7,15,30 and 60 days after surgery.The rabbits in control group group were sacrified for immunohistological study after bred for 30 days.The expressions of glial fibrillary acidic protein (GFAP) and brain derived neurotrophic facor (BDNF) were observed.Results In operation group, the outer nulear layer of retina thinned, and the cells in the inner nulear layer was disorganized 7,15,and 30 days after the surgery;glial cells proliferated 60 days after surgery; the positive expression of BDNF and GFAP was more than that in the shamoperation and control group.In shamoperation group, the positive expression of BDNF and GFAP was more than that in the control group.No obvious difference of expression of BDNF and GFAP between each time point groups was found.Conclusions The expression of neroprotective related proteins increased after subretinal implantation with inactive chips suggests that limited neuroprotective effects might be led by the implantation.
Optic nerve glioma (ONG) is a rare central nervous system tumor that occurs in children and adolescents. It’s main pathological type is low-grade pilocytic astrocytoma. It is divided into sporadic ONG and neurofibromatosis type 1 (NF-1) related ONG. Due to the close relationship between ONG and the optic nerve, there is its particularity in diagnosis and treatment. The diagnosis of ONG mainly relies on medical history, symptoms and signs, as well as imaging examinations such as MRI and CT. ONG should be differentiated from optic nerve sheath meningioma, optic neuritis, optic nerve metastasis and other diseases. In recent years, newly discovered molecular targeted therapy and anti-vascular endothelial growth factor drugs are a powerful supplement to ONG. When chemotherapy is not sensitive or resistant, radiotherapy can be considered, but it is only recommended for patients over 7 years of age. Surgery can be considered when the patient’s visual impairment is severe and the appearance of the eye is significantly affected. In addition, due to the susceptibility of NF-1 patients to tumors, the chemotherapy regimen should take into account the risk of secondary leukemia caused by the drug, and the timing of radiotherapy should be after the age of 10. We look forward to further ONG clinical research, which will bring more references for future clinical work.
Ischemic retinopathy, resulting in multiple lesions like microvasculature damage, inflammation and neovascularization, is a major contributor of vision damage. In these pathological changes, retinal glia cannot be ignored in the development of retinopathy. They constitute a highly versatile population that interacts with various cells to maintain homeostasis and limit disease. Therefore, glial activation and gliosis are strikingly ubiquitous responses to almost every form of retinal disease. Both of microglial cells and Müller cells are major intrinsic retinal glial cells and they are in close relationship, which means they can influence each other, make joint action or even become interdependent. They exhibit morphological and functional changes to have an impact on degree of retinal injury through different responses, which mediated by glial cells are important not only for course of disease progression, but also for the maintenance of neuronal and photoreceptor survival. Thus, defining the mechanisms that underlie communications between microglial cells and Müller cells could enable the development of more selective therapeutic targets, with great potential clinical applications.
Objective To observe the dynamic expression of nestin and glial fibrilary acidic protein (GFAP) in the development of retina in rats.Methods In 48 Wistar rats, 24 were divided into 8 groups with 3 rats in each according to their age (1 day, 1 week, and 2, 3, 4, 7, 12, and 20 weeks old). The sagittal freezing sections of the eye were made; nestin/glutamine synthetase (GS) and GFAP/GS were stained by immunofluorescence and were observed under the confocal microscope. Total RNA was extracted from 18 rats which were divided into 6 groups according to the age (1 day, 1 week, and 2, 3, 4, and 12 weeks old) with 3 rats in each. The expression of nestin, GAFA and GS mRNA were detected by realtime quantitative reverse transcription polymerase chain reaction (RT-PCR). Müller cells were cultured from postnatal day 7-12 rats; the expression of nestin and GFAP was detected by immunostaining study. Double immunofluorescence was carried out between nestin/GS and GFAP/GS.Results One day after the birth, nestin positive cells were found in the whole retinal neuroblast layers with elongated retinal progenitor cells; the GFAP positive astrocytes were observed in the inner retina. One week after the birth, Müller glial cells expressed GS and nestin but not GFAP; GFAP positive cells localized in the inner retina.Two to 12 weeks after the birth, the expression of nestin in Müller cells decreased and even disappeared; the expression of GFAP in astrocytes didn't change much. The Müller cells expressed nestin but no GFAP in vitro. The expression of nestin and GFAP mRNA in retina was accordant with the results of immunofluorescence staining.Conclusion In the developing retina, the expression of nestin in Müller cells decreases gradually, and no expression of nestin can be found in adult rats; the expression of GFAP can't be observed in Müller cells in neonatal and adult 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.
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.