Fig. 8
Proof of concept experiment investigating the impact of overexpressing GR (gene ID: Nr3c1) in Müller cells of the diabetic mouse retina in vivo. A Experimental design and the time line of treatment and readouts in relation to the development of diabetes in the db/db mice. B 2.5 × 1013 GC/ml AAV9-Nr3c1-EGFP particles were injected intravitreally in 3-month-old diabetic and control mice. Tissue was collected 3 months thereafter and stained for the Müller cell marker glutamine synthetase (GLUL). EGFP indicated successful viral transduction. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bars, 20 µm. C qPCR on MACS-sorted retinal cells isolated from 6-month-old mice, 3 months after single AAV injection, was performed. Only Müller cell-specific gene expression is shown. Bars represent mean values ± SEM. Biological replicates were as follows: n = 7 in controls and n = 5 for db/db mice. Unpaired t-test: *p < 0.05. D Assessment of microglial responses upon AAV-treatment reveals reduced microglial activation in retinae with Müller glia-specific GR overexpression. Left, Representative micrographs depicting the Iba1-staining in retinal flat mounts that were used to assess microglial morphological alterations. Right, Quantification of microglial cell numbers (bars represent the mean ± SEM from 2 mice per genotype and condition), soma size and the area occupied by their processes. For the latter two parameters, bars represent the mean ± SEM: n = 30 microglia in control retinae with PBS-sham injection; n = 102 microglia in db/db retinae with PBS-sham injection; n = 99 microglia in db/db retinae with AAV injection. These cells were analysed in retinal flatmounts from two mice per genotype and condition. The bigger the soma area and the smaller the occupied area, the more activated the microglia are. Ordinary one-way ANOVA with Dunnett's multiple comparisons test, ***p < 0.001. E Electroretinogram recordings were performed 3 months after AAV injection. Primarily rod (scotopic)- or cone-driven responses (photopic) or mixed responses were quantified. A-wave (reflects photoreceptor responses) and b-wave (representing inner retinal response e.g. by bipolar cells) were evaluated. Bars represent mean ± SEM of 5 animals per genotype. The right eye received the AAV injection, while the contralateral eye was injected with an equal volume of PBS as sham control. In both control and db/db animals, one PBS-sham-injected eye had to be excluded from analysis because of cataract formation or intraocular haemorrhage after surgery, resulting in the following number of biological replicates per genotype and treatment: n = 4 PBS-sham-injected eyes from control mice; n = 5 AAV-injected eyes from control mice; n = 4 PBS-sham-injected eyes from db/db animals; n = 5 AAV-injected eyes from db/db mice. Unpaired t-test: *p < 0.05