Abstract
Purpose :
Aniridia often results in progressive corneal clouding termed aniridia-related keratopathy (ARK), but the causes of this corneal clouding are not well understood. The current study investigates three hypotheses as to the causes of corneal clouding in ARK: decreased ocular surface integrity, increased conjunctivalization, and activation of the pro-fibrotic response. Clinical, histological, and immunofluorescent evaluations were performed in the Pax6+/- mouse model Small Eye (Sey).
Methods :
Wild-type (Pax6+/+) and Sey (Pax6+/-) corneas were assessed at two time points: 17-19 weeks old (mild ARK symptoms) (N=10 wild type, 6 Sey) and 40+ weeks old (severe ARK symptoms)(N=6 wild type, 6 Sey). Clinical assessment was performed by corneal clarity and ocular surface integrity scoring via fluorescein uptake.Histological assessment was performed on one eye of each mouse via paraffin sectioning, hematoxylin and eosin staining and periodic acid-schiff (PAS) staining to assess conjunctivalization. Two-way analysis of variance was performed for statistical analysis of PAS staining. Immunofluorescent evaluation was performed on one eye of each mouse via cryosectioning, blocking with 0.2% Bovine Serum Albumin and 5% goat serum, and incubating in 1:1000 dilution primary antibodies a-SMA (myfibroblast marker) and collagen type III (marker of fibrotic wound healing response).
Results :
Consistent with our hypothesis, as corneal clarity decreased, ocular surface integrity decreased in Sey mice at each time point. Two way ANOVA showed average number of conjunctivalization in mouse corneas differed between wild-type and Sey mice (df=1, F=10.367, p<0.01). Conjunctivalization did not differ between 17-19 week old and 40+ week old mice (df=1, F=3.163, p=0.106). As corneal clarity decreased in 40+ week old Sey mice as compared to 17-19 week old Sey and wild type, this finding was contrary to our hypothesis. Our hypothesis of activation of the pro-fibrotic response was confirmed through immunofluorescent evaluation, with expression of a-SMA and collagen type III in the anterior stroma as corneal clouding progressed.
Conclusions :
We have furthered the hypotheses of ocular surface integrity and activation of the pro-fibrotic response contributing to progressive corneal clouding in ARK, while our hypothesis of increased conjunctivalization was not supported. The findings of pro-fibrosis could open pathways for potential treatment modalities.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.