Figure 4.

Primary ONHA and TMC were treated for 48 hours with various concentrations of minocycline only or additionally treated with 600 μM H₂O₂ for four hours. After exposure of the cells to the substance alone and in combination with oxidative stress, viability was determined by staining all nuclei with Hoechst 33342 and dead cells with propidium iodide. (A) Representative fluorescence photomicrograph of Hoechst 33342-stained, untreated ONHA as control. (B) Nonviable cells in the corresponding field. Fluorescence photomicrograph of ONHA treated with minocycline 20 μM (C) and 40 μM (E) for 48 hours and labeled with Hoechst 33342. Nonviable ONHA treated with minocycline 20 μM (D) and 40 μM (F) for 48 hours in the same field as in (C) and (E). Fluorescence photomicrograph of ONHA treated with 600 μM H₂O₂ only (G) and with minocycline 20 μM (I) and 40 μM (L) for 48 hours and additionally treated with 600 μM H₂O₂ and labelled with Hoechst 33342. Nonviable ONHA treated with 600 μM H₂O₂ only (H) and with minocycline 20 μM (K) and 40 μM (M) for 48 hours and additionally treated with 600 μM H₂O₂ in the same field as in (G, I, L). TMC yielded a similar result (data not shown). Quantification of effect of minocycline treatment alone and additional treatment with H₂O₂ on numbers of nonviable primary ONHA (N) and TMC (O). The percentage of dead cells was scored by counting at least 500 cells in fluorescence photomicrographs of representative fields. Data (mean±SD) are based on the sampling of 6–10 photomicrographs per condition in three independent experiments performed in duplicate. Comparison with controls is shown above the bars with significant differences marked by an asterisk.

Abbreviations: TMC, trabecular meshwork cells; ONHA; optic nerve head astrocytes; SD, standard deviation.