Fig. 1
Characterisation of the phenotype of the db/db (Leprdb) mice as an animal model for DR in T2D. A Timeline of the development of features of T2D and DR in the db/db mouse model. Time points of data collection are highlighted as dots on the time line and respective readouts are listed in the boxes above. B Left, representative micrographs of PDGFRβ staining to delineate pericytes (yellow arrows) in retinal flatmount preparations of diabetic and control mice 9 months of age. Scale bar, 20 µm. Right, PDGFRβ-positive cells were counted per scan field (410 µm × 298 µm) of both genotypes. Bars represent mean ± SEM and comprise data from the following number of biological replicates: 3 months of age, n = 4 mice per genotype; 6 months of age, n = 6 mice per genotype; 9 months of age, n = 3 mice per genotype.. Unpaired t-test: *p<0.05. C Left, representative micrograph of Iba1 stainings of retinal flat mounts from 6-month-old diabetic and control mice. Scale bar, 20 µm. Right, Iba1-positive cells were quantified per scan field (410 µm × 298 µm). Z-scans through the whole thickness of the retina were performed and cells across all retinal layers were counted. In addition, the soma area of each microglia in a scan field was measured as an indicator of beginning microglial activation. Bars represent mean ± SEM and comprise data from the following number of biological replicates: 3 months of age, n = 4 mice per genotype; 6 months of age, n = 6 mice per genotype; 9 months of age, n = 3 mice per genotype. Unpaired t-test: *p<0.05. D GFAP was only present in astrocytes residing in the nerve fibre layer, but not in Müller cells—neither in healthy controls nor in retinae from db/db mice 6-months of age. E Patch clamp recordings of isolated Müller cells from 6-month-old db/db mice demonstrated a significant reduction of amplitudes known to be primarily mediated by Kir4.1 potassium channels. Bars represent mean ± SD (standard deviation) and comprise data from ~ 40 cells collected from 4 animals per genotype. Mann–Whitney-test: ***p<0.01. F The ability of Müller cells to compensate for hypoosmotic stress was tested in the retina of 6-month-old db/db mice. Vital retinal sections were exposed to hypoosmolar stress (60% osmolarity) for 4 minutes. Changes in the Müller cell soma area, visualised by labelling with Mitotracker Orange, were measured as an indication of cell swelling. Bars represent mean ± SEM and comprise data from ~15 cells collected from 2 animals per genotype. Mann–Whitney-test: *p < 0.05.