Fig 2: Cyclosporine A facilitates corneal epithelium wound healing and improves the light transmission of the cornea in Cisd2KO mice. (a) Representative photographs for the corneal wound healing of Cisd2KO and WT mice with cyclosporine A (CsA) or normal saline (NS) treatment. Cisd2KO and WT mice at 3-mo are divided into 4 groups, namely WT-CsA, WT-NS, Cisd2 KO-CsA, and Cisd2KO-NS. A 1.5-mm diameter corneal epithelial defect is made in the right eye using an Alger Brush under anesthesia; the left eye serves as an un-injured control. Cyclosporine A (0.05%) or normal saline (0.9% NaCl) 3 times a day are applied to the injured eyes till the end of the study. In the WT-NS and WT-CsA groups, the corneal wounds are almost completely healed by day 7 after wounding. However, in the Cisd2KO-NS group, delayed wound healing is noted; intriguingly, CsA treatment facilitates the epithelial wound healing in the Cisd2KO-CsA group. (b) Quantification of unhealed wound surface in WT and Cisd2KO mice with CsA or NS treatment (n=3). The injured corneas are stained with 1% fluorescein and photographed with slit lamp every 24 hours for 17 days after the injury, and the size of the unhealed wound surface areas are measured and analyzed by a computer-assisted digitizer (Image J). (c) Quantification of light transmission of the corneas in WT and Cisd2KO mice with CsA or NS treatment (n=3). Light transmission assay is carried out to evaluate the functional improvement by CsA. Immediately after corneal wounding study, the mice were sacrificed and the trephined corneoscleral buttons with endothelial side up are processed for transmission spectrophotometer. The transmittance percentage of the Cisd2 KO-NS group is significantly lower than that in the WT-NS and WT-CsA groups. Remarkably, in the Cisd2 KO-CsA group, the transmittance percentage is significantly higher than that in Cisd2 KO-NS group. Data are presented as mean ± SD. ***p<0.001. In (b) and (c), the statistical analysis was performed by generalized equation estimation (GEE). (d) Pathological analysis reveals that CsA treatment ameliorates abnormal corneal manifestations and inflammation in the Cisd2KO-CsA group. In both the WT-NS and WT-CsA groups, the corneas remained clear and fluorescein showed negative staining after complete re-epithelialization. In addition, the normal stratification of corneal epithelia and regular alignment of corneal stromal collagen layers are well demonstrated by Masson's trichrome staining; furthermore, CD45-positive cell is not observed in WT cornea. However, in the Cisd2KO-NS group, exaggerated epithelial thickening, abnormal epithelial stratification and loss of stromal collagen rectangular alignment with cellular infiltration were noted. Notably, in the Cisd2KO-CsA group, the abnormal corneal manifestations are ameliorated by CsA treatment; moreover, CD45-positive cell is dramatically diminished after applying CsA.