Corneal immune cell activation and development of keratopathy in type 1 and type 2 murine models of diabetes mellitus

Purpose : Most patients with long-term diabetes mellitus (DM) present with some degree of keratopathy (DK), but despite high incidence, its underlying inflammatory mechanism is not yet elucidated. Here we investigate the impact of immune cell activation on corneal nerve damage and its relationship to the clinical manifestations of DK. Methods : Type 1 DM (T1D) was induced in 6-week-old C57BL/6 mice with streptozotocin. 4-week-old B6.Cg-Lep ob/J mice were used to model type 2 DM (T2D). Non-DM (ND) mice were used as controls (n=12/group/timepoint). Mice were followed for 12 weeks and clinical assessments (in vivo confocal microscopy (IVCM), optical coherence tomography (OCT), corneal fluorescein staining (CFS), phenol red thread test, Cochet-Bonnet esthesiometry) were performed once a week. Corneas were harvested (4 and 12 weeks) and either fixed for immunostaining and confocal imaging or digested. Cell suspensions were analyzed by flow cytometry or FACS- sorted and assessed by RT-PCR or ELISA. Results : After 4 weeks of disease induction, 1) total frequencies of CD45 CD11b Ly- 6G- myeloid cells were higher in both T1D (3.2%, p=0.006) and T2D (3.5%, p=0.0016) compared to ND (1.4%), 2) there were enhanced levels of gene expression of the pro-inflammatory cytokines TNF-α and IL-1β in both T1D (IL-1β p=0.0002, TNF-α p<0.0001) and T2D (IL-1β p=0.0005, TNF-α p<0.0001) compared to ND and 3) the number of myeloid cells/mm2 in the sub-basal neural plexus (SBNP) was significantly higher in T1D (46.7, p<0.0001) and T2D (56.7, p<0.0001) compared to ND (15.38). DK clinical manifestations started at 4 weeks with significantly lower 1) tear production in T1D (p=0.0001) and T2D (p<0.0001); 2) corneal sensitivity in T1D (p=0.404) and T2D (p=0.0038); and 3) CFS scores significantly higher T1D mice (p<0.0001) and T2D (p<0.0001), all compared to ND. At 12 weeks, nerve density in the SBNP was lower in both T1D (p=0.001) and T2D (p=0.002) compared to ND. Intraepithelial terminal endings per 40X field were lower in T1D (p<0.001) and T2D (p<0.001) compared to ND. Epitheliopathy was confirmed in DM mice by IVCM. Conclusions : Murine models of T1D and T2D demonstrate comparable changes in immune cell frequency and activation, SBNP and intraepithelial nerve damage, epitheliopathy, and clinical manifestations; therefore, it can be a powerful tool to further study the immune mechanism of DK.