Fig 1.
(A) Example of a whole mount, 18 hours post-wound, stained with Ly6G (green) to visualize infiltrating neutrophils and DAPI (blue). Radial cuts divide the cornea into four petals, enabling the cornea to flatten under the coverslip. The wound area is visible in the central cornea surrounded by the parawound region (yellow). The location of the original wound margin is demarcated (red). Scale bar = 100μm (B) Each cornea was measured from the limbus across the center to the opposite limbal region using x- and y- image coordinates and images were taken at even intervals, designated by points 1 through 4.
Table 1.
Primer sequences for real-time PCR.
Fig 2.
In vivo wound closure is delayed with vitamin D treatment.
Mice were wounded and treated with vehicle (0.02% ethanol/PBS) or 1,25D3 (10-7M) every 6 hours or left untreated (control) for 24 hours. (A) Corneal wound areas were monitored by fluorescein staining every 6 hours. (B) Wound area remaining open was determined as a percentage of original wound area. Data represent mean ± SEM and were analyzed with two-way repeated measures ANOVA and Bonferroni’s correction for multiple comparisons. * = p<0.05 (comparison between vehicle and vitamin D treatments; n = 9 mice/group).
Fig 3.
Basal epithelial cell division across the wounded cornea at 18 hours after wounding.
Nuclei of basal epithelial cells were identified with DAPI staining. The graph represents the average number of mitotic nuclei counted per field in vehicle or 1,25D3 treated corneas 18 hours after wounding. (n = 5 corneas/group).
Fig 4.
Vitamin D treatment increases neutrophil infiltration in wounded corneas.
(A) Total neutrophil counts across the cornea at 18 hours post-wound from fields 1 through 4. Data represent mean ± SEM and were analyzed with Student’s two-tailed t-test, p<0.01 (n = 6). (B) Neutrophil counts per field in vehicle or vitamin D-treated corneas at 18 hours after wounding. Statistical analysis was by two way ANOVA with Bonferroni’s test for multiple comparisons. p<**0.01, ****<0.0001 (n = 6).
Fig 5.
Chemokine CXCL1 and CXCL2 expression in wounded corneas.
CXCL1 (A) and CXCL2 (B) expression was determined in corneal homogenates following 12 and 24 hours of wounding. For protein analysis (top row), 8 corneas were isolated per time point per treatment group for each experiment, were pooled, and total corneal protein was collected for ELISA analysis of chemokine expression. Graphs are representative data from one experiment (n = 2, 24hrs; n = 1, 12hrs) showing the mean and ± SD of duplicate technical repeats. For RNA analysis (bottom row), relative gene expression was determined by RT-PCR analysis. 10 individual corneas were isolated per time point per treatment group for each experiment. Graphs are representative data from one experiment (n = 3, 24hrs; n = 1, 12hrs) showing the mean ± SEM of triplicate technical repeats.
Fig 6.
Topical vitamin D treatment does not change subbasal nerve density or VEGF expression following wounding.
(A) Corneal whole mounts were stained with anti-tubulin β III and subbasal nerves counted in a 10x10 morphometric grid to determine relative nerve densities. The graph represents the average nerve density per field in vehicle or 1,25D3 treated corneas 18 hours after wounding. (n = 5 corneas/group). (B) VEGF expression was determined in corneal homogenates 12 and 24 hours after wounding. For RNA analysis (left), relative expression was determined by RT-PCR analysis. Samples represent 10 pooled corneas per group at each time point. Graphs are representative data from one experiment (n = 3, 24hrs; n = 1, 12hrs) showing the mean ± SEM of triplicate values. For protein analysis (right), total corneal protein was collected and ELISA performed. Data represent 8 pooled corneas per group at each time point. Graphs are representative data from one experiment (n = 2, 24hrs; n = 1, 12hrs) showing the mean of duplicate values.
Fig 7.
Cytokine expression at 12 and 24 hours after corneal wounding.
TNFα, IL-1β, TGFβ1, and TGFβ2 expression was determined in corneal homogenates following 12 and 24 hours of wounding by RT-PCR. Samples represent 10 pooled corneas per group at each time point. Graphs are representative data from one experiment (n = 3, 24hrs; n = 1, 12hrs) showing the mean ± SEM of triplicate values.
Fig 8.
Vitamin D increases the expression of CRAMP in wounded corneas.
CRAMP expression was determined in corneal homogenates following 12 and 24 hours of wounding by RT PCR. 10 individual corneas were isolated and pooled per time point per treatment group for each experiment. Graphs are representative data from one experiment (n = 2, 24hrs; n = 1, 12hrs) showing the mean ± SEM of triplicate technical repeats.
Fig 9.
Topical vitamin D treatment does not affect wound closure rate in a human organ culture wounding model but wounded corneas express a higher level of the vitamin D activating enzyme, CYP27B1.
(A, B) Corneal wounds were monitored by fluorescein staining every 6 hours for 24 hours. Data were analyzed with repeated measures ANOVA and Bonferroni’s correction for multiple comparisons. (n = 5) (C) Corneal epithelium was collected at the time of wounding (initial) and after 48 hours of regrowth (regrown) and CYP27B1 expression was determined by RT-PCR. Data were analyzed by Student’s two-tailed t-test. * = p<0.05 (n = 4).