RESUMO
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.
RESUMO
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 combina-tion 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 ana-lyzing 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 pre-vious studies were not reliable.