**3. Results**

#### *3.1. Topical Administration of GLP-1 has no E*ff*ect on Body Weight and Systemic Blood Glucose Levels*

No significant difference was observed in body weight and blood glucose concentrations during the experiment between db/db mice treated with GLP-1 eye drops and db/db mice treated with vehicle (Figure 1A,B).

**Figure 1.** Evolution of (**A**) body weight and (**B**) blood glucose levels in the experimental groups.

#### *3.2. Topical Administration of GLP-1 Reduces DNA*/*RNA Damage through the Decrease of Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS) Induced by Diabetes in the Retina*

The impaired equilibrium between ROS and the antioxidant defenses promotes oxidative stress that affects the structure of several molecules, including nucleic acids. The hydroxyl radicals can damage DNA by converting deoxyguanosine into 8-hydroxyguanosine. Here we provide evidence that this phenomenon occurred in an experimental model of DR (db/db mice) and that the topical administration of GLP-1 could prevented this process (Figure 2A,B).

**Figure 2.** Immunofluorescence analysis of DNA/RNA damage (8-hydroxiguanosine) and nitro tyrosine. (**A**,**B**) Comparison and quantification of 8-hydroxiguanosine (red) protein levels through immunofluorescence among representative samples of diabetic retinas treated with vehicle eye drops (black bars) or GLP-1 eye drops (gray bars) and non-diabetic retinas (white bars). Hoechst staining (blue) was used for nuclei labeling. Optical magnifications of the ganglion cell layer (GCL) and the inner nuclear layer (INL) are also displayed. Scale bars, 30 μm. *n* = 4; (**C**,**D**) comparison and quantification of nitro tyrosine (red) protein levels through immunofluorescence among representative samples of diabetic retinas treated with vehicle eye drops (black bars) or GLP-1 eye drops (gray bars) and non-diabetic retinas (white bars). Hoechst staining (blue) used for nuclei labeling. Scale bars, 30 μm. *n* = 4; \* *p* < 0.05. GCL—ganglion cell layer; INL—inner nuclear layer; IPL—inner plexiform layer; ONL—outernuclearlayer;OPL—outerplexiformlayer.

RNS act similar to ROS in terms of cell damage. In fact, nitro tyrosine protein levels were also increased in the retinas of diabetic mice in comparison with non-diabetic mice. GLP-1 significantly reduced them too (Figure 2C,D).

*3.3. GLP-1 Eyedrops Protect from Oxidative Stress by Increasing the Protein Levels of Glutathione Reductase, Glutathione Peroxidase and Copper–Zinc and Manganese Superoxide Dismutases (CuZnSOD and MnSOD) in Diabetic Retinas*

Glutathione (GSH) e ffectively scavenges free radicals and other ROS and RNS (e.g., hydroxyl radical, lipid peroxyl radical, superoxide anion and hydrogen peroxide) directly and indirectly through enzymatic reactions. The reduced GSH can be regenerated from oxidized GSH by glutathione redox cycle. However, in the diabetic retina, the enzymes responsible for glutathione redox cycle (glutathione peroxidase and glutathione reductase) are compromised [6,8,10]. We observed a statistically insignificant increase of protein levels of glutathione peroxidase (Figure 3A,B) and glutathione reductase (Figure 3C,D) in diabetic retinas treated with GLP-1 eye drops in comparison with those treated with vehicle.

**Figure 3.** Protein levels of glutathione peroxidase and glutathione reductase. ( **A**,**B**) Densitometric analysis and western blot bands of glutathione peroxidase corresponding to retinas of db/db mice treated with vehicle eye drops (black bars), GLP-1 eye drops (gray bars) and to non-diabetic mice retinas (white bars). Protein levels normalized with cyclophilin A. *n* = 3; ( **C**,**D**) densitometric analysis and western blot bands of glutathione reductase corresponding to retinas of db/db mice treated with vehicle eye drops (black bars), GLP-1 eye drops (gray bars) and to non-diabetic mice retinas (white bars). Protein levels normalized with cyclophilin A. *n* = 3; \* *p* < 0.05.

Ultimately, the radical chain reactions will be blocked by the antioxidant enzymes superoxide dismutase (SOD). The activities of both CuZnSOD, located in the cytosol, and MnSOD in the mitochondria are decreased in diabetic retina [6,8,10]. In the present study, we found that CuZnSOD and MnSOD levels were significantly increased by the topical administration of GLP-1 (Figure 4A–D and Figure 4E–H, respectively); (*p* < 0.05).

**Figure 4.** Protein levels of copper–zinc and manganese superoxide dismutase (CuZnSOD and MnSOD) (**A**,**B**) Comparison and quantification of CuZnSOD (green) protein levels through immunofluorescence among representative samples of diabetic retinas treated with vehicle eye drops (black bars) or GLP-1 eye drops (gray bars) and non-diabetic retinas (white bars). Hoechst staining (blue) used for nuclei labeling. GCL—ganglion cell layer; INL—inner nuclear layer; IPL—inner plexiform layer; ONL—outer nuclear layer; OPL—outer plexiform layer. Scale bars, 20 μm. *n* = 4; (**C**,**D**) densitometric analysis and western blot bands of CuZnSOD corresponding to retinas of db/db mice treated with vehicle eye drops (black bars), GLP-1 eye drops (gray bars) and to non-diabetic mice retinas (white bars). Protein levels normalized with vinculin. *n* = 3; (**E**,**F**) comparison and quantification of MnSOD (green) protein levels through immunofluorescence among representative samples of diabetic retinas treated with vehicle eye drops (black bars) or GLP-1 eye drops (gray bars) and non-diabetic retinas (white bars). Hoechst staining (blue) used for nuclei labeling. Scale bars, 20 μm. *n* = 4; (**G**,**H**) densitometric analysis and western blot bands of MnSOD corresponding to retinas of db/db mice treated with vehicle eye drops (black bars), GLP-1 eye drops (gray bars) and to non-diabetic mice retinas (white bars). Protein levels normalized with vinculin. *n* = 3; \* *p* < 0.05. GCL—ganglion cell layer; INL—inner nuclear layer; IPL—inner plexiform layer; ONL—outer nuclear layer; OPL—outer plexiform layer.

#### *3.4. Topical Administration of GLP-1 Activates the Expression in the Retina of Proteins Involved in DNA Repair (Babam2) and Cell Proliferation (Ki67)*

Reactive oxygen and nitrogen species damage cellular macromolecules including DNA. Babam2 or BRE (brain and reproductive organ-expressed protein) is part of the BRCA1, a complex which is implicated in both DNA repair and maintenance of G2/M arrest in reaction to DNA damage [11,12]. For this reason, we wanted to assess Babam2 in our experiment. We found that the retina of untreated diabetic mice had considerably increased DNA/RNA damage compared with controls and that treatment with GLP-1 eye drops significantly increased the protein levels of Babam2 in retina in diabetic mice (Figure 5A–C). Moreover, GLP-1 increases Ki67 protein levels in neuroretina and favors its translocation to the nucleus, thus indicating the promotion of neurogenesis in the diabetic retina (Figure 6A,B).

**Figure 5.** Babam2 protein levels. (**A**) Comparison of colabelling immunofluorescence assay for Babam2 (red) with NeuN (neuronal specific marker) (green) in db/db mice among representative samples of diabetic retinas treated with vehicle eye drops or GLP-1 eye drops and non-diabetic retinas. Nuclei labeled with Hoechst stain nuclei specific marker) (blue). GCL—ganglion cell layer; INL—inner nuclear layer; IPL—inner plexiform layer; ONL—outer nuclear layer; OPL—outer plexiform layer. An orthogonal view of Babam2 to analyze nuclear translocation in GCL of db/db mice treated with vehicle, db/bb mice treated with GLP-1 eye drops and non-diabetic mice are also displayed in this figure. Scale bars, 30 μm. *n* = 4; (**B**,**C**) densitometric analysis and western blot bands of babam2 corresponding to retinas of db/db mice treated with vehicle eye drops (black bars), GLP-1 eye drops (gray bars) and to non-diabetic mice retinas (white bars). Protein levels normalized with Gadph. *n* = 3; \* *p* < 0.05. GCL—ganglion cell layer; INL—inner nuclear layer; IPL—inner plexiform layer; ONL—outer nuclear layer; OPL—outer plexiform layer.

**Figure 6.** (**A**) Comparison of ki67 (red) protein levels through immunofluorescence among representative samples of diabetic retinas treated with vehicle eye drops or GLP-1 eye drops and non-diabetic retinas. Ki67 is colabelled with NeuN (neuronal specific marker) (green) and Hoechst staining (nuclei specific marker) (blue); (**B**) Optical magnifications of GCL and INL are also presented in this figure. Scale bars, 30 μm. *n* = 4. GCL—ganglion cell layer; INL—inner nuclear layer; IPL—inner plexiform layer; ONL—outer nuclear layer; OPL—outer plexiform layer.
