Corticosteroids effects on keratocytes under normal culture conditions.

<p>(A)Morphological results of keratocytes after 5-d treatments with PBS, PA, Dex and Flu. (B)Proliferation in each group was determined by CCK8 assayed at 12 h, and then every 24 h during 5 days. (C)Short-term cell viabilities in each group were tested by CCK8 at 12 h (a) and 24 h (b). (D)The migration rates of keratocytes at 12 h(a) and 24 h(b) were evaluated by Image Pro-Plus software (Media Cybernetics) using the following formula: (0 h distance—set time distance)/ time (Pixel/h). (E)Migration abilities of keratocytes grown in normal condition with corticosteroid treatments at 0 h, 12 h and 24 h. Data were analyzed by one way ANOVA,*<i>p</i><0.05 versus control group (n = 3).</p

Corticosteroids effects on Col I and Col VI expression.

<p>Immunofluorescence staining was carried out on control and corticosteroids-treated keratocytes after 48 h treatment. (A, B) Staining result of Col I (green) was shown, and corresponding fluorescence intensity in each group was also quantified. (C, D) Col VI immunofluorescence staining results were shown (red), and the result was further examined by western blot and corresponding grey scale scanning. Slides were mounted with Hoechst 33528. Data were expressed in mean ± SD, analyzed by one way ANOVA, *<i>p</i><0.05 versus control group (n = 3).</p

Effect of corticosteroids on the release of pro-inflammatory cytokines and MMP9 in LPS pretreated keratocytes.

<p>Keratocytes were treated with 10 μg/ml LPS to induce an inflammatory response. Corticosteroid-LPS groups were added LPS and corticosteroids at the same time to see corticosertoids’ effects on keratocytes under inflammatory condition. The levels of TNF-α (A), IL-6 (B) and IL-1β (C) were evaluated after 48 h in culture. (D)Western blot result and corresponding grey value scanning of MMP9 expression was shown. *<i>p</i><0.05 versus control group, #<i>p</i><0.05 versus LPS group (n = 3).</p

Corticosteroids effects on Col I, VI secretions under inflammatory condition.

<p>(A, C)The control and LPS keratocytes cells with PA, Dex, or Flu treatments were stained with the Col I (green), VI (red) marker. (B) Quantified result of Col I immunofluorescence. (D) Col VI expressions by Western blotting and its quantification results. Slides were mounted with Hoechst 33528. Data were expressed in mean ± SD, analyzed by one way ANOVA, *<i>p</i><0.05 versus control group, #<i>p</i><0.05 versus LPS group (n = 3).</p

Representative images from transmission electron microscopy showing the conjunctival epithelial ultrastructure.

<p>Compared with normal eyes, fewer goblet cells and secretary granules were observed on day 0 in Groups BAC-W2 and BAC-W3, and on days 0, 7, and 21 in Group BAC-W4. This loss of goblet cells and secretary granules was particularly pronounced in Group BAC-W5 on all of the days that were studied. Chromatin condensation and peripheral migration could be observed on days 0, 7, and 21 in Group BAC-W5.</p

The results of Schirmer's tests in all groups.

<p>Schirmer's test results show decreased aqueous tear secretion compared to pre-treatment secretion levels on days 0, 7 and 21 after the cessation of BAC treatment. *P<0.05, **P<0.01, ***P<0.001, respectively. Compared with pre-treatment, the differences were statistically significant on days 0 and 7 in the BAC-W2, BAC-W3, and BAC-W4 groups as well as on days 0, 7 and 14 in Group BAC-W5. The values shown are average wetted lengths ± standard deviations.</p

Representative images from transmission electron microscopy.

<p>Compared with images from normal eyes, swelling of the mitochondria, Golgi apparati and RER could be observed on day 0 in Group BAC-W2, on days 0 and 7 in Groups BAC-W3 and BAC-W4, and on days 0, 7, and 21 in Group BAC-W5.</p

Representative images from transmission electron microscopy that revealed the ultrastructure of the corneal epithelium.

<p>Compared with normal eyes, vacuoles between the cells could be observed on day 0 in Group BAC-W2 and on days 0, 7, and 21 in Groups BAC-W3, BAC-W4. These vacuoles were particularly prominent among samples from Group BAC-W5.</p

The scores for sodium fluorescein staining in all groups.

<p>Compared with the pre-treatment group, there were significant differences on days 0, 7 and 14 after the cessation of BAC treatment in the BAC-W2, BAC-W3, BAC-W4, and BAC-W5 groups.</p

Representative pictures of HE staining of the corneas from each of the four groups.

<p>Normal corneas had three to five epithelial layers. Damage to the epithelium could be observed on days 0 and 7 in the four groups.</p