- Schirmer test values
There were no significant differences among the four groups at baseline, on day 3 and 7.(Table 3)
- Rose Bengal staining
The Rose Bengal staining scores differed among the four groups on day 0, 3, and 7 (on day 0; 0.08 ± 0.12 in the control group, 1.56 ± 0.60 in the 1-minute PI group, 3.22 ± 0.46 in the 3-minutes PI group, and 5.56 ± 0.56 in the 10-minutes PI group; on day 3; 0.33 ± 0.33 in the control group, 1.44 ± 0.60 in the 1-minute PI group, 3.00 ± 0.58 in the 3-minutes PI group, and 4.00 ± 0.47 in the 10-minutes PI group; on day 7; 0.00 ± 0.00 in the control group, 0.89 ± 0.26 in the 1-minute PI group, 1.89 ± 0.59 in the 3-minutes PI group, and 3.56 ± 0.78 in the 10-minutes PI group, Figure 1). However, significant differences were observed between the control and 3-minutes PI group, between the 3-minutes and 10-minutes PI groups, and between the 1-minute and 10-minutes PI groups only immediately after instillation (p = 0.042, p = 0.040, p = 0.016, respectively, according to the Steel–Dwass test), and no significant differences were observed between the control and the 1-minute PI group, and between the 1-minute and the 3-minutes PI group (p = 0.155, p = 0.582, respectively, according to the Steel–Dwass test). There were no significant differences between the four groups on day 3 and 7 (p > 0.05 in the Steel–Dwass test). Overall, the area stained with Rose Bengal tended to increase as PI exposure time increased. And the Rose Bengal staining showed a tendency to improve with passage of time in all PI groups. However, only the 10-minutes PI group was statistically significant (p = 0.607 in control group, p = 0.905 in 1-minute PI group, p = 0.513 in 3-minutes PI group, and p = 0.04 in 10-minutes PI group by Friedman’s test).
- Corneal fluorescein staining
The corneal fluorescein staining scores were counted of 0.5, 5.5, 5.0 and 8.5 each in the control group, 1-minute, 3-minutes and 10-minutes PI group, respectively on day 0, 0.25, 2.5, 2.75 and 7.5 respectively on day 3, and 0.25, 1.5, 2.5 and 7.25 respectively on day 7 (Figure 2). On day 3, some eyes of the 10-minutes PI group showed large epithelial defect on superficial cornea and no improvement until day 7 (Figure 2d). The corneal fluorescein staining score showed significant improve with passage of time only in the 1-minute and 3-minutes PI groups (p = 0.607 in control group, p = 0.038 in 1-minute PI group, p = 0.022 in 3-minutes PI group, and p = 0.584 in 10-minutes PI group by Friedman’s test). The control and three PI groups showed no significant differences in the corneal fluorescein staining score at each time point (p > 0.05 by the Steel Dwass test). However, there were obvious corneal epithelial damages on some eyes of the 10-minutes PI group compared with the other groups.
- Conjunctival impression cytology
1) Goblet cell density (GCD)
The baseline GCD in the conjunctiva, before any instillation, was 92.3 ± 15 in the control group, 88.6 ± 11.2 in the 1-minute PI group, 92.4 ± 13.9 in the 3-minutes PI group, and 93.7 ± 8.3 in the 10-minutes PI group. And when the GCD of the four groups were compared immediately after the PI instillation (on day 0), there were no significant differences between the four groups (p values of Tukey-Kramer test were 0.971, 0.996, 0.998, 0.976, 0.931 and 0.663 in the control vs 1-minute PI group, control vs 3-minutes PI group, control vs 10-minutes PI group, 1-minute vs 3-minutes PI group, 1-minute vs 10-minutes PI group, 3-minutes vs 10-minutes PI group, respectively).
However, there were apparent significant differences on day 3 and 7 after PI instillation between the control and 3-minutes PI group, the control and 10-minutes PI group, the 1-minute and 3-minutes PI group, the 1-minute and 10-minutes PI group, and the 3-minutes and 10-minutes PI group (all p < 0.001 in the Tukey–Kramer test), whereas the control and 1-minute PI group were not different significantly (p = 0.560 on day 3, p = 0.868 on day 7 in the Tukey–Kramer test). On day 3, the GCD was 87.7 ± 7.4 in the control group, 80.9 ± 7.4 in the 1-minute PI group, 30.5 ± 11.2 in the 3-minutes PI group, and 10.3 ± 6.2 in the 10-minutes PI group. On day 7, the GCD was 76.9 ± 8.2 in the control group, 80.1 ± 10.8 in the 1-minute PI group, 50.2 ± 12.1 in the 3-minutes PI group, and 8.6 ± 8.8 in the 10-minutes PI group (Figure 3).
2) Severity of squamous metaplasia noted by conjunctival impression cytology
When the severity of squamous metaplasia was compared using the Nelson grading system, the Nelson score tended to increase with PI exposure time on day 3 and 7. The Nelson score of the conjunctiva was 0.00 ± 0.00 in all group on day 0. On day 3, the Nelson score of the conjunctiva was 0.20 ± 0.30 in the control group, 0.87 ± 0.20 in the 1-minute PI group, 1.70 ± 0.30 in the 3-minutes PI group, and 2.20 ± 0.40 in the 10-minutes PI group. On day 7, the Nelson score of the conjunctiva was 0.33 ± 0.30 in the control group, 0.70 ± 0.40 in the 1-minute PI group, 1.80 ± 0.40 in the 3-minutes PI group, and 2.40 ± 0.45 in the 10-minutes PI group. The Nelson score was significantly different between the 1-minute and 3-minutes PI group and between the 1-minute and 10-minutes PI groups on day 7 (p = 0.013, p = 0.0031 by Steel–Dwass test), whereas no significant difference was observed between the control and 1-minute PI group (p = 0.766). And this score showed a tendency to improve with passage of time in the 1-minute, 3-minutes, and 10-minutes PI groups (p = 0.012, p = 0.001, p < 0.001 by Friedman’s test) (Figure 4).
5. Light microscopic examination of conjunctival tissue
Conjunctival cells prepared on day 7 were observed by light microscopy. Abundant goblet cells and multiple layers of epithelial cells were apparent in the control and 1-minute PI group. However, the goblet cell counts decreased, and single layers of epithelial cells became more prevalent as the PI exposure time increased to more than 3 minutes (Figure 3). These findings were similar to those obtained upon conjunctival impression cytology test.
6. Immunofluorescence staining for MUC5AC of conjunctival tissue
Goblet cells were observed by immunofluorescence staining using an anti-MUC5AC antibody. Abundant MUC5AC was present in the conjunctival epithelia of the control and 1-minute group. However, the extent of MUC5AC staining was reduced markedly in the 3-minutes and 10-minutes PI groups compared to the other groups (Figure 3).
7. Transmission electron microscopy (TEM) of conjunctival tissue
Under the transmission electron microscope (TEM), abundant microvillar structures were evident on the surfaces of conjunctival epithelial cells of the control and 1-minute PI group. In addition, goblet cells with many secretory granules, which play an important role in synthesis of MUC5AC, were observed (Figure 5a, 5b). However in both the 3-minutes and 10-minutes PI groups, the extent of microvillar structure decreased, and apoptotic morphological changes including nuclear fragmentation, condensation and peripheral migration of chromatin were evident in conjunctival epithelial cells (Figure 5c, 5d). Such ultrastructural disturbances were more severe in the 10-minutes PI group (Figure 5d).
- Scanning electron microscopy (SEM) of corneal tissues
Scanning electron microscopy (SEM) showed that the superficial cells were intact with normal microvilli in the control group (Figure 5e). However, SEM images showed corneal epithelial toxicity that the microvilli seemed to be injured in the all three PI groups. The longer the exposure time, the greater was the reduction in the number of microvilli (Figure 5f, 5g, 5h).