In order to study the in vitro culture and expansion of bone marrow mesenchymal stem cells in rats (rMSCs) and the possibility of rMSCs differentiation into retinal neural cells, the bone marrow-derived cells in SD rats were isolated and cultured in vitro. The retinal neural cells in SD rats were cultured and the supernatants were collected to prepare conditioned medium. The cultured rMSCs were induced to differentiate by two steps. Immunofluorescence method and anti-nestin, anti-NeuN, anti-GFAP and anti-Thy1.1 antibodies were used to identify the cells derived from the rMSCs. The results showed that the in vitro cultured rMSCs grew well and expanded quickly. After induction with two conditioned media, rMSCs was induced to differentiate into neural progenitor cells, then into retinal neural-like cells which were positive for nestin, NeuN, GFAP and Thy1.1 detected by fluorescence method. The findings suggested that rMSCs could be culture and expanded in vitro, and induced to differentiate into retinal neural-like cells.
Animals, Newborn ; Bone Marrow Cells/*cytology ; Cell Differentiation/*physiology ; Cells, Cultured ; Mesenchymal Stem Cells/*cytology ; Neurons/*cytology ; Rats, Sprague-Dawley ; Retina/*cytology

In the vertebrate retina, Müller cells are principal glial cells which stretch across the whole thickness of the retina and contact with the somata and processes of all retinal neurons, thus forming an anatomical and functional link between glial cells and retinal neurons. Numerous studies have shown that Müller cells express various neurotransmitter receptors, transporters, ion channels and enzymes that are relative to cellular activities. In addition, the cells also release factors, such as D-serine and glutamate etc., to regulate the neuron excitability. Therefore, retinal Müller cells may play more curious roles in addition to supporting the retinal neurons. The information exchange and interaction between Müller cells and neurons may regulate and maintain retinal neuronal functions. In the glaucomatous retina, Müller cells are reactivated (gliosis). Reactivated Müller cells undergo a variety of changes in cellular physiology, biochemistry and morphological features. Meanwhile, the reactivated Müller cells may produce and release cytotoxic factors, such as nitric oxide (NO), tumor necrosis factor-α (TNF-α), reactive oxygen species (ROS) and prostaglandin E2 (PGE2), thus involving in the induction of retinal ganglion cell apoptosis and death. Here, we reviewed the physiological properties of retinal Müller cells, and the functional changes of Müller cells in the glaucomatous retina.
Ependymoglial Cells ; pathology ; physiology ; Glaucoma ; physiopathology ; Humans ; Neurons ; physiology ; Retina ; cytology

The electrophysiological effects of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (NMPTP), a chemical inducer of Parkinsonism in man and monkey, on the pigmented rabbit retina were determined under acute conditions. The amplitude of the b-wave of the rabbit electroretinogram was affected, but both the implirit time and half-amplitude duration of it werenot. The amplitude of the photopic b-wave was increased by 72.9 +/- 32.1% 5hours after the intravenous injection of NMPTP (P[t] < 0.05), whereas that of the scotopic b-wave was decreased by 31.2 +/- 6.4% 4hours after injection (P[t] < 0.05). The above results suggest or support that: 1, the dopaminergic amacrine cells are related to the modulation of the b-wave of the rabbit electroretinogram. 2. during light adaptation, the dopaminergic amacrine cells uncouple the rod and cone systems in the inner plexiform layer and are involved in functions of the rod system. 3. the hypothesis that the funrtion of tyrosine hydroxylase may be affected by NMPTP.
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine ; Animals ; Electrophysiology ; *Electroretinography ; Pyridines/*pharmacology ; Rabbits ; Retina/cytology/*drug effects

BACKGROUNDEndothelial progenitor cells (EPCs) transplantation is a promising therapeutic strategy for ischemic retinopathy. The current study aimed to establish a simple, reliable and fluorescent labeling method for tracking EPCs with 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) in laser-injured mouse retina.

METHODSEPCs were isolated from human umbilical cord blood mononuclear cells, cultivated, and labeled with various concentrations of CFSE. Based on fluorescence intensity and cell morphology, a 15 minutes incubation with 5 µmol/L CFSE at 37°C was selected as the optimal labeling condition. The survival capability and the apoptosis rate of CFSE-labeled EPCs were measured by Trypan blue staining and Annexin V/PI staining assay respectively. Fluorescence microscopy was used to observe the label stability during the extended culture period. Labeled EPCs were transplanted into the vitreous cavity of pigmented mice injured by retinal laser photocoagulation. Evans Blue angiography and flat mounted retinas were examined to track the labeled cells.

RESULTSEPCs labeled with 5 µmol/L CFSE presented an intense green fluorescence and maintained normal morphology, with no significant changes in the survival capability or apoptosis rate after being labeled for 2 days, 1 and 4 weeks. The fluorescence intensity gradually decreased in the cells at the end of 4 weeks. Evans Blue angiography of the retina displayed the retinal capillarity network clearly and fluorescence leakage was observed around photocoagulated spots in the laser-injured mouse model. One week after transplantation of labeled EPCs, the fluorescent cells were identified around the photocoagulated lesions. Four weeks after transplantation, fluorescent tube-like structures were observed in the retinal vascular networks.

CONCLUSIONEPCs could be labeled by CFSE in vitro and monitored in vivo for at least 4 weeks, and participate in the repair of injured retinal vessels.

Animals ; Cells, Cultured ; Endothelial Cells ; chemistry ; cytology ; Fluoresceins ; chemistry ; Fluorescent Dyes ; chemistry ; Humans ; Male ; Mice ; Mice, Inbred C57BL ; Microscopy, Fluorescence ; Retina ; cytology ; Stem Cells ; chemistry ; cytology ; Succinimides ; chemistry

PURPOSE: To investigate the asymmetry of the retinal nerve fiber layer thickness (RNFLT) with respect to the horizontal and vertical meridian and between the right and left eye in normal subjects. METHODS: The RNFLT was measured in 121 normal volunteers by optical coherence tomography (OCT). The RNFLT was analyzed by dividing the circle scanning area (diameter 3.4 mm) around the optic disc into 4 quadrants and 12 sectors. RESULTS: There was a significant difference between the RNFLT of the nasal and temporal quadrant in individual eyes. There was a significant difference between the RNFLT of corresponding sectors with respect to the vertical or horizontal meridian in individual eyes. The nasal and temporal RNFLTs were asymmetrical between the right and left eye in the quadrant and sector analysis. The RNFLT of the nasal and temporal quadrant was thicker in the right eye. The nasal and inferior RNFLT measured by OCT had a significant correlation with degree of refractive error. CONCLUSIONS: In normal subjects without significant anisometropia, there was significant asymmetry of the RNFLT for each eye as well as between the right and left eye.
*Tomography, Optical Coherence ; Retinal Ganglion Cells/*ultrastructure ; Retina/*cytology ; Reference Values ; Optic Disk/cytology ; Nerve Fibers/*ultrastructure ; Male ; Humans ; Female ; Adult

BrdU (5-Bromo-2'-deoxyuridine) is usually used to label the mitotic cells as well as to trace reagent in cell transplation. However, BrdU could also exert some side effect on cellular biological characteristics upon inappropriate use. To explore the appropriate concentration of BrdU for labelling retinal progenitor cells (RPCs), we co-cultured Embryonic day (E) 17. 5 RPCs with different concentrations of BrdU, which were 0.2, 1, 5 and 10 micromol/L, respectively. After 48 hours, the RPCs were proliferation- or differentiation-cultured. Immunofluorescence was used to detect the BrdU-positive ratio and differentiation potential. Cell count was used to evaluate proliferation ability, and lactate dehydrogenase (LDH) release assay was used to monitor cytotoxicity. The results showed that 0.2 micromol/ L BrdU could not label RPCs clearly, while BrdU of 1, 5 or 10 micromol/L could label the RPCs with similar ratios. 1 micromol/L BrdU displayed no obvious cytotoxicity and showed no obvious effect on the proliferation and differentiation ability. However, 5 micromol/L or 10 micromol/L BrdU could evidently inhibit RPCs proliferation, partly due to the cytotoxicity effect. Furthermore, 10 micromol/L BrdU could inhibit the differentiation of RPCs towards MAP2-positive nerve cells, but showed no influence on the differentiation of RPCs towards GFAP- and glutamine synthetase positive glial cells. This study suggested that 1 micromol/L BrdU could be an appropriate concentration for RPCs labelling and could efficiently label RPCs without obvious side effect.
Animals ; Bromodeoxyuridine ; chemistry ; Cell Culture Techniques ; methods ; Cell Differentiation ; Cell Proliferation ; Embryo, Mammalian ; Immunohistochemistry ; Mice ; Retina ; cytology ; Staining and Labeling ; Stem Cells ; cytology

OBJECTIVETo examine the change of Smoothened (Smo) expression in the retinofugal pathway and in the growth cones during the period of embryonic day 13 (E13) to E15.

METHODSSmo expression in the chiasm and growth cones was observed by fluorescent immunostaining and retinal explant culture.

RESULTSOn E13 and E14, Smo was expressed moderately in the retina and optic disc, and in the corner of the retina, Smo expression was especially dense. On E13, Smo expression was detected in the optic nerves and ventral diencephalon, but only in the superficial region of the optic tract on E14. Smo was also detected in the stem and filopodia of the growth cones in the retinal explant culture during this period.

CONCLUSIONSmo expression changes in different developmental phases, suggesting that Smo might play a role in signal optic axon growth during the development of the retinofugal pathway.

Animals ; Fluorescent Antibody Technique ; Mice ; Mice, Inbred C57BL ; Optic Chiasm ; cytology ; embryology ; metabolism ; Optic Nerve ; cytology ; embryology ; metabolism ; Receptors, G-Protein-Coupled ; biosynthesis ; Retina ; cytology ; embryology ; metabolism ; Smoothened Receptor ; Visual Pathways ; cytology ; embryology ; metabolism

BACKGROUNDNeural stem cells (NSCs) transplantation and gene therapy have been widely investigated for treating the cerebullar and myelonic injuries, however, studies on the ophthalmology are rare. The aim of this study was to investigate the migration and differentiation of brain-derived neurotrophic factor (BDNF) gene transgenic NSCs transplanted into the normal rat retinas.

METHODSNSCs were cultured and purified in vitro and infected with recombinant retrovirus pLXSN-BDNF and pLXSN respectively, to obtain the BDNF overexpressed NSCs (BDNF-NSCs) and control cells (p-NSCs). The expression of BDNF genes in two transgenic NSCs and untreated NSCs were measured by fluorescent quantitative polymerase chain reaction (FQ-PCR) and enzyme-linked immunosorbent assay (ELISA). BDNF-NSCs and NSCs were infected with adeno-associated viruses-enhanced green fluorescent protein (AAV-EGFP) to track them in vivo and served as donor cells for transplantation into the subretinal space of normal rat retinas, phosphated buffer solution (PBS) served as pseudo transplantation for a negative control. Survival, migration, and differentiation of donor cells in host retinas were observed and analyzed with Heidelberg retina angiograph (HRA) and immunohistochemistry, respectively.

RESULTSNSCs were purified successfully by limiting dilution assay. The expression of BDNF gene in BDNF-NSCs was the highest among three groups both at mRNA level tested by FQ-PCR (P < 0.05) and at protein level measured by ELISA (P < 0.05), which showed that BDNF was overexpressed in BDNF-NSCs. The results of HRA demonstrated that graft cells could survive well and migrate into the host retinas, while the immunohistochemical analysis revealed that transplanted BDNF-NSCs differentiated into neuron more efficiently compared with the control NSCs 2 months after transplantation.

CONCLUSIONSThe seed cells of NSCs highly secreting BDNF were established. BDNF can promote NSCs to migrate and differentiate into neural cells in the normal host retinas.

Animals ; Brain-Derived Neurotrophic Factor ; genetics ; metabolism ; Cell Differentiation ; physiology ; Cell Movement ; physiology ; Cells, Cultured ; Embryo, Mammalian ; cytology ; Enzyme-Linked Immunosorbent Assay ; Immunohistochemistry ; Neurons ; cytology ; Rats ; Retina ; cytology ; metabolism ; Stem Cell Transplantation

OBJECTIVETo establish the inner blood-retinal barrier (BRB) model in vitro by co-culturing RF/6A cells and C6 cells and to investigate the effects of EMP (200 kV/m, 200 pulses) exposure on the permeability of the inner BRB model in vitro.

METHODSRF/6A cells and C6 cells were co-cultured on transwell, and the characteristic of the inner BRB model was assessed by detecting transendothelial electrical resistance (TEER) and the permeability of horseradish peroxidase (HRP). The co-cultured model was exposed or sham exposed to the EMP (200 kV/m 200 pulses) for 0.5, 3, 6, 12, 24 h in vitro, then TEER and the permeability of HRP were measured for studying the effects of EMP on the permeability of inner BRB model in vitro.

RESULTSTEER value (145 Ωcm(2)) of the co-culturing inner BRB model significantly increased, as compared to that of RF/6A cells alone model (P < 0.05) on the 6th day after inoculation. There was significant difference of permeability of HRP between the co-culturing inner BRB model and RF/6A cells alone model (P < 0.05). The ability of inhibiting large molecular materials in the co-culturing inner BRB model enhanced. The TEER value decreased and the permeability of HRP increased as compared to the sham group at 0.5, 3, 6 h after the exposure.

CONCLUSIONThe inner BRB model by co-culturing RF/6A cells and C6 cells in vitro is efficient and suitable to study the alterations of the restricted permeability function of the inner BRB. EMP (200 kV/m for 200 pulses) could induce the enhanced permeability of the inner BRB model in vitro.

Animals ; Blood-Retinal Barrier ; physiology ; Cell Line, Tumor ; Coculture Techniques ; Electric Impedance ; Electromagnetic Fields ; Endothelial Cells ; physiology ; Macaca mulatta ; Permeability ; Rats ; Retina ; cytology

The present study was undertaken to determine the effect of epigallocatechin gallate (EGCG) on lipopolysaccharide (LPS)-induced production of inflammatory chemokine regulated upon activation normal T cell expressed and secreted (RANTES) in human retinal endothelial cells (HRECs) and to explore the underlying regulatory mechanism. HRECs were stimulated with LPS in the presence or absence of EGCG at various concentrations (100, 50, 25, 12.5, 6.25 μmol/L). The optimum concentration of drug was determined by a real-time cell-electronic sensing (RT-CES) system, and MTS chromatometry was used to detect the toxicity of LPS and EGCG on HRECs. RANTES production in the culture supernatant was measured by ELISA. The expression levels of Akt and phosphorylated Akt were examined by Western blot assay. The result showed that LPS markedly stimulated RANTES secretion from HRECs. EGCG treatment significantly suppressed LPS-induced RANTES secretion in a dose-dependent manner. Furthermore, EGCG exhibited a dose-dependent inhibitory effect on LPS-induced phosphorylation of Akt. Taken together, our data suggest that EGCG suppresses LPS-induced RANTES secretion, possibly via inhibiting Akt phosphorylation in HRECs.
Catechin ; analogs & derivatives ; pharmacology ; Cells, Cultured ; Chemokine CCL5 ; metabolism ; Endothelial Cells ; metabolism ; Humans ; Lipopolysaccharides ; Phosphorylation ; Proto-Oncogene Proteins c-akt ; metabolism ; Retina ; cytology