ABSTRACT
This study aimed to modify the mixed and purified culture of rat retinal ganglion cells (RGCs) in vitro. The retinae of 1-3 day old Sprague-Dawley (SD) rats were separated bluntly into two layers: inner layer and outer layer, under a surgical microscope. Retinal cells isolated from different layers (inner layer, outer layer and whole retinal tissue) by using enzyme dissociation method were cultured in F12/DMEM medium containing 15% FBS. After 3-day culture, the RGCs in the retinal cells obtained from mixed culture of inner, outer, and whole retinal tissue were identified by immunocytochemical staining of Thy-1.1, and the rate of RGCs to retinal cells (RGCs%) was calculated. Two monoclonal antibodies, anti-macrophages/granulocytes (OX-41) against rat macrophage and antibody against rat Thy-1.1 (OX-7), were used to purify RGCs by either a conventional or modified two-stepped immunopanning procedure (purification in situ). Purified RGCs were seeded at different cell density and cultured in F12/DMEM medium containing 15% FBS. Immunocytochemical staining for Thy-1.1, MTT, and PI-Hoechst33342 fluorescence imaging were used to identify the purity and the viability of RGCs in purified culture of RGCs. The results showed: (1) Immunocytochemistry of different retinal tissue layers culture revealed that the RGCs% was (19.9±1.2)%, (0.5±0.2)%, and (6.2±1.7)% respectively in the mixed culture of inner, outer, and whole retinal tissue, with differences being significant (P<0.05); (2) fluorescent double staining of Hoechst33342 and PI indicated that with the same RGCs%, RGCs obtained from purification in situ grew well with more neurite outgrowth than those by the conventional two-stepped immunopanning method; (3) the viability of purified RGCs seeded at high density was increased and the cells developed complex intercellular networks. The viability of RGCs was declined with the decreasing seeding density, and most cells presented round or oval in shape with thin neurites. It was concluded that: (1) RGCs% in the inner layer retina was higher than that in the outer layer retina; (2) RGCs obtained by in situ purification had more neurite outgrowth and lower mortality than those by conventional two-stepped immunopanning procedure; (3) the viability of purified RGCs could be increased by increasing cell seeding density to some extent.
ABSTRACT
Clopidogrel was believed to be superior to aspirin by the well-known CAPRIE trial. However, no other large clinical trials demonstrated the same results, but all focused on the combination use of clopidogrel with aspirin, and combination therapy in CREDO was called the "Emperor's New Clothes". However, no one overturned the results of these clinical trials by quantitatively analyzing them. We reviewed ten large-scale clinical trials about clopidogrel. On the basis of results of CAPRIE, CREDO and CHARISMA trials, we re-estimated their minimal sample sizes and their powers by three well-established statistical methodologies. From the results of CAPRIE, we inferred that the minimal sample size should be 85 086 or 84 968 but its power was only 30.70%. A huge gap existed. The same was also true of CREDO and CHARISMA trials. Moreover, in CAPRIE trial, 0 was included in the 95% confidence interval and 1 was included in the 95% confidence interval for the relative risk. There were some paradoxical data in CAPRIE trial. We are led to conclude that the results in CAPRIE, CREDO, and from the subgroup analysis in CHARISMA trials were questionable. These results failed to demonstrate that clopidogrel was superior to aspirin or that clopidogrel used in combination with aspirin was better than aspirin alone. The cost-effectiveness analyses by some previous studies were not reliable.
ABSTRACT
To explore the biological effects of light on trabecular cells, cultured bovine trabecular cells were exposed to visible light of different wavelength with different energy. Cellular morphology, structure, proliferation, and phagocytosis were observed. The cells showed no remarkable changes when the energy was low. When the exposure energy reached 1.12 mW/cm2, the cytoplasm showed a rough appearance, and cell proliferation and phagocytosis decreased. This phototoxicity was strong with white light (compound chromatic light), moderate with violet light or yellow light, and mild with red light.
Subject(s)
Animals , Cattle , Cell Proliferation , Radiation Effects , Cells, Cultured , Light , Phagocytosis , Radiation Effects , Trabecular Meshwork , Cell Biology , Radiation EffectsABSTRACT
To explore the biological effects of light on trabecular cells, cultured bovine trabecular cells were exposed to visible light of different wavelength with different energy. Cellular morphology, structure, proliferation, and phagocytosis were observed. The cells showed no remarkable changes when the energy was low. When the exposure energy reached 1.12 mW/cm2, the cytoplasm showed a rough appearance, and cell proliferation and phagocytosis decreased. This phototoxicity was strong with white light (compound chromatic light), moderate with violet light or yellow light, and mild with red light.
Subject(s)
Cell Proliferation/radiation effects , Cells, Cultured , Light , Phagocytosis/radiation effects , Trabecular Meshwork/cytology , Trabecular Meshwork/radiation effectsABSTRACT
To investigate whether the TGF-beta 1 plasmid DNA carried by lipofectamine could be introduced into cultured rabbit corneal epithelial cells, specific expression of the plasmid pMAM TGF-beta 1 in the cultured corneal epithelial cells was studied. Two days after 12 h of transfection of pMAMT-GF-beta 1 mediated by lipofectamine into the cultured corneal epithelial cells, the TGF-beta 1 protein expression specific for pMAMTGF-beta 1 in the cells was detected by means of immunohistochemical staining and the positive rate was 23.37%. The results suggested that foreign plasmid DNA could be effectively delivered into cultured rabbit corneal epithelial cells by means of lipofectamine, and this will provide a promising method of studying TGF-beta 1 on the mechanism of physiology and pathology concerned with corneal epithelial cells.
Subject(s)
Animals , Rabbits , Cell Division , Cells, Cultured , Epithelium, Corneal , Cell Biology , Metabolism , Gene Transfer Techniques , Genetic Vectors , Lipids , Pharmacology , Plasmids , Genetics , Transforming Growth Factor beta , Genetics , Transforming Growth Factor beta1ABSTRACT
The method of gene transfer into corneal endothelium was investigated to provide a foundation for the study of TGF-beta 1 gene transfer to inhibit corneal graft rejection. Two days after direct injection of pMAM TGF-beta 1 mediated by liposome into the anterior chamber of rabbits, one half of corneas were made into paraffin slides and the endothelial layer was carefully torn from the other half to make a single layer slide of endothelia. By means of immunohistochemical technique, the plasmid pMAM TGF-beta 1 expression product TGF-beta 1 in the endothelia was detected. Specific TGF-beta 1 expression was positive in the endothelia on both the paraffin slide and the single layer slide. The results showed that by direct injection into the anterior chamber, foreign plasmid DNA could be transferred into the endothelia and its expression was obtained. This may provide a foundation for further study on TGF-beta 1 participating in local induction of corneal immune tolerance.
Subject(s)
Animals , Rabbits , Anterior Chamber , Corneal Transplantation , Endothelium, Corneal , Pathology , Gene Transfer Techniques , Immune Tolerance , Transforming Growth Factor beta , GeneticsABSTRACT
The method of gene transfer into corneal endothelium was investigated to provide a foundation for the study of TGF-beta 1 gene transfer to inhibit corneal graft rejection. Two days after direct injection of pMAM TGF-beta 1 mediated by liposome into the anterior chamber of rabbits, one half of corneas were made into paraffin slides and the endothelial layer was carefully torn from the other half to make a single layer slide of endothelia. By means of immunohistochemical technique, the plasmid pMAM TGF-beta 1 expression product TGF-beta 1 in the endothelia was detected. Specific TGF-beta 1 expression was positive in the endothelia on both the paraffin slide and the single layer slide. The results showed that by direct injection into the anterior chamber, foreign plasmid DNA could be transferred into the endothelia and its expression was obtained. This may provide a foundation for further study on TGF-beta 1 participating in local induction of corneal immune tolerance.
Subject(s)
Anterior Chamber , Corneal Transplantation , Endothelium, Corneal/drug effects , Endothelium, Corneal/pathology , Gene Transfer Techniques , Immune Tolerance , Transforming Growth Factor betaABSTRACT
In order to explore whether the conventional use of 5-fluorouracil (5-Fu) had any toxic effects on trabecular meshwork cells, bovine trabecular meshwork cells were cultured in vitro and exposed to 5-Fu at different concentrations. The cellular morphology, ultrastructure, mortality and phagocytosis were studied under light microscopy, transmission electron microscopy and methods of Wright's stain. It was found that the toxic effects of 5-Fu on the cells were in a dose-dependent mode. 1×10-1 mg/ml of 5-Fu caused a large part of cells rounded up, while 1×10-3 mg/ml of the drug only a rough appearance of the cell surface. Exposure to 1×10-2 mg/ml of 5-Fu made mitochrone swollen and rough endoplasmic reticulum enlarged, with the cell mortality being 50.5 %. The latex microspheres engulfed in cytoplasm in cells receiving 1×10-1 and 1×10-2 mg/ml of 5-Fu were significantly decreased as compared with those in the control group (P<0.01). It was concluded that the safe concentration of 5-Fu on bovine trabecular meshwork cells was 1×10-3 mg/ml and the conventional dosage of 5-Fu in clinical practice would not cause injury to trabecular meshwork cells.
ABSTRACT
In order to explore whether the conventional use of 5-fluorouracil (5-Fu) had any toxic effects on trabecular meshwork cells, bovine trabecular meshwork cells were cultured in vitro and exposed to 5-Fu at different concentrations. The cellular morphology, ultrastructure, mortality and phagocytosis were studied under light microscopy, transmission electron microscopy and methods of Wright's stain. It was found that the toxic effects of 5-Fu on the cells were in a dose-dependent mode. 1×10-1 mg/ml of 5-Fu caused a large part of cells rounded up, while 1×10-3 mg/ml of the drug only a rough appearance of the cell surface. Exposure to 1×10-2 mg/ml of 5-Fu made mitochrone swollen and rough endoplasmic reticulum enlarged, with the cell mortality being 50.5 %. The latex microspheres engulfed in cytoplasm in cells receiving 1×10-1 and 1×10-2 mg/ml of 5-Fu were significantly decreased as compared with those in the control group (P<0.01). It was concluded that the safe concentration of 5-Fu on bovine trabecular meshwork cells was 1×10-3 mg/ml and the conventional dosage of 5-Fu in clinical practice would not cause injury to trabecular meshwork cells.
ABSTRACT
Objective To observe the effect of superoxide dismutase(SOD)-compound on bacterial ulceration in rabbit's eyes and explore its mechanism.Methods Amikacin and SOD-compound etc.were added to agar inoculating PS.Pyocyaneus and the diameter of inhabiting circles were measured.The rabbit model of bacterial corneal ulcer was created and then treated with amikacin.SOD-compound was used as an adjunctive treatment while inactive SOD-compound as a control.The clinical manefestation and its pathological change were observed and the amlonyldialdehyde (MDA) in the ulcer was assayed.Results In vitro experiment indicated that SOD-compound had no anti-bacterial effect.In rabbit corneal ulcer treated with SOD-compound,the clinical manefestation and pathological change was slighter,the MDA was lower.Conclusion In rabbit's corneal ulceration,free radical and lipid peroxidation take part in the tissue damage.SOD-compound can contradict this effect through eliminating excessive free radical and anti-lipid peroxidation.Therefore,SOD-compound has protective effect on corneal physiology and histological structure in bacterial corneal ulceration.