Ing was higher in Tet-mev-1/Dox(+) mice compared with that in the other three types of mice (Fig. 3b). The corneal fluorescein staining scores were 0.7560.89, 1.1460.90, 0.7161.25, and 4.5061.60 for WT/Dox(2) mice, Tet-mev-1/ Dox(2) mice, WT/Dox(+) mice, and Tet-mev-1/Dox(+) mice (all n = 8), respectively. The score ranged from 0 to 9 with a score of 0 indicating normal and a score of 9 indicating a severe cornealpunctuate MK 8931 defect. The corneal fluorescein staining score for Tetmev-1/Dox(+) mice was significantly worse than for the other three types of mice (n = 8, ANOVA Tukey’s test, p,0.00001) (Fig. 3c).DiscussionIt is well known that lacrimal and salivary gland functions decline with age in humans [33,34]. We first hypothesized that both lacrimal and salivary gland functions decline in Tet-mev-1/ Dox(+) mice. However, the severe inflammation and fibrosis associated with functional decline occurred in the lacrimal gland, but not in the salivary gland. We hypothesized that the inherent 
tissue responses to oxidative stress in the lacrimal and salivary glands are different. Pharmacological 370-86-5 cholinergic blockade (subcutaneous injection of scopolamine hydrobromide) inhibits lacrimal gland function. It also stimulates inflammatory cytokine production and lymphocytic infiltration in the lacrimal gland. This systemic cholinergic blockade does not induce a nonspecific inflammation at three sites (conjunctival goblet cells, submandibular glands and small intestine) that receive cholinergic innerva-Figure 2. Lacrimal gland in Tet-mev-1 mice with Dox has functional depression of mitochondria and excessive O22production. A, The activity of complexes I and II in WT/Dox(+) vs. Tet-mev-1 mice/Dox(+). NADH-cytochrome c oxidoreductase was applied as an enzymatic indicator of 18055761 complex I activity, and succinate-coenzyme Q oxidoreductase as an enzymatic indicator of complex II activity. Although there were no differences in the activity of complex I between these mice, complex II was significantly decreased in Tet-mev-1 mice with Dox. (WT: n = 5, Tet-mev-1: n = 3, NS, not significant; *P,0.01 [Student’s t-test]). The vertical bars indicate the standard deviation of the separate experiments. B, Production of O22 in the lacrimal gland was significantly increased in Tet-mev-1/Dox(+) compared with that in the other types of mice. (n 5, *P = 0.0014 [Kruskal-Wallis test]). The vertical bars indicate the standard deviation of the separate experiments. C, Carbonyl protein content of the lacrimal gland by ELISA. Each value shows the ratio of Tet-mev-1 and WT for the relative amount of carbonyl protein in Tet-mev-1 mice with or without Dox (n = 4, *P = 0.004 [Student’s ttest]). D, Immunohistochemical staining of 8-OHdG: Tet-mev-1/Dox(+) shows more positive nuclei (brown, indicated by the arrow) than the other types of mice. doi:10.1371/journal.pone.0045805.gOxidative Stress Induced Dry Eye DiseaseFigure 3. Tet-mev-1/Dox(+) have dry eye disease. A, Aqueous tear production: Aqueous tear quantity values of Tet-mev-1/Dox(+) were significantly lower than those in the other types of mice (n 6, ANOVA Tukey’s test, p = 0.0024). B, Tet-mev-1/Dox(+) mice had more corneal fluorescein staining than in the other mice. C, The corneal fluorescein staining score of Tet-mev-1/Dox(+) was significantly worse than that in the other types of mice (all n = 8, ANOVA Tukey’s test, p,0.00001). doi:10.1371/journal.pone.0045805.gtions [35]. These results suggest that the lacrimal gland is subjec.Ing was higher in Tet-mev-1/Dox(+) mice compared with that in the other three types of mice (Fig. 3b). The corneal fluorescein staining scores were 0.7560.89, 1.1460.90, 0.7161.25, and 4.5061.60 for WT/Dox(2) mice, Tet-mev-1/ Dox(2) mice, WT/Dox(+) mice, and Tet-mev-1/Dox(+) mice (all n = 8), respectively. The score ranged from 0 to 9 with a score of 0 indicating normal and a score of 9 indicating a severe cornealpunctuate defect. The corneal fluorescein staining score for Tetmev-1/Dox(+) mice was significantly worse than for the other three types of mice (n = 8, ANOVA Tukey’s test, p,0.00001) (Fig. 3c).DiscussionIt is well known that lacrimal and salivary gland functions decline with age in humans [33,34]. We first hypothesized that both lacrimal and salivary gland functions decline in Tet-mev-1/ Dox(+) mice. However, the severe inflammation and fibrosis associated with functional decline occurred in the lacrimal gland, but not in the salivary gland. We hypothesized that the inherent tissue responses to oxidative stress in the lacrimal and salivary glands are different. Pharmacological cholinergic blockade (subcutaneous injection of scopolamine hydrobromide) inhibits lacrimal gland function. It also stimulates inflammatory cytokine production and lymphocytic infiltration in the lacrimal gland. This systemic cholinergic blockade does not induce a nonspecific inflammation at three sites (conjunctival goblet cells, submandibular glands and small intestine) that receive cholinergic innerva-Figure 2. Lacrimal gland in Tet-mev-1 mice with Dox has functional depression of mitochondria and excessive O22production. A, The activity of complexes I and II in WT/Dox(+) vs. Tet-mev-1 mice/Dox(+). NADH-cytochrome c oxidoreductase was applied as an enzymatic indicator of 18055761 complex I activity, and succinate-coenzyme Q oxidoreductase as an enzymatic indicator of complex II activity. Although there were no differences in the activity of complex I between these mice, complex II was significantly decreased in Tet-mev-1 mice with Dox. (WT: n = 5, Tet-mev-1: n = 3, NS, not significant; *P,0.01 [Student’s t-test]). The vertical bars indicate the standard deviation of the separate experiments. B, Production of O22 in the lacrimal gland was significantly increased in Tet-mev-1/Dox(+) compared with that in the other types of mice. (n 5, *P = 0.0014 [Kruskal-Wallis test]). The vertical bars indicate the standard deviation of the separate experiments. C, Carbonyl protein 
content of the lacrimal gland by ELISA. Each value shows the ratio of Tet-mev-1 and WT for the relative amount of carbonyl protein in Tet-mev-1 mice with or without Dox (n = 4, *P = 0.004 [Student’s ttest]). D, Immunohistochemical staining of 8-OHdG: Tet-mev-1/Dox(+) shows more positive nuclei (brown, indicated by the arrow) than the other types of mice. doi:10.1371/journal.pone.0045805.gOxidative Stress Induced Dry Eye DiseaseFigure 3. Tet-mev-1/Dox(+) have dry eye disease. A, Aqueous tear production: Aqueous tear quantity values of Tet-mev-1/Dox(+) were significantly lower than those in the other types of mice (n 6, ANOVA Tukey’s test, p = 0.0024). B, Tet-mev-1/Dox(+) mice had more corneal fluorescein staining than in the other mice. C, The corneal fluorescein staining score of Tet-mev-1/Dox(+) was significantly worse than that in the other types of mice (all n = 8, ANOVA Tukey’s test, p,0.00001). doi:10.1371/journal.pone.0045805.gtions [35]. These results suggest that the lacrimal gland is subjec.
