**4. Discussion**

DR physiopathology embraces several metabolic pathways triggered by the hyperglycemic state. One of them is the overproduction of free radicals and the consequent nitro-oxidative imbalance that leads to cell damage [5]. GLP-1 and GLP-1R agonists have emerged as potential drugs against DR due to their neuroprotective properties, but their e ffect on DR oxidative stress has not been assessed [1,3,13]. Herein, we provide evidence that an antioxidant e ffect can be added to the underlying mechanism by which GLP-1R agonists exert their beneficial action on the diabetic retina.

In the present study, we found diabetes-induced oxidative stress resulting in DNA/RNA damage through guanine oxidation and protein damage by tyrosine nitration. In this regard, retinas of diabetic mice presented higher levels of both markers (8-hydroxy-2--deoxyguanosine and nitrotyrosine, respectively) in comparison with non-diabetic mice, which implies the appearance of nitro-oxidative stress as a consequence of the DR state. Notably, these deleterious e ffects were prevented by the topical administration of GLP-1. Several studies have reported the relationship between GLP-1 and the decrease of cellular ROS levels [14]. Wang et al. showed that GLP-1 could reduce high-glucose-induced ROS in cardiac microvascular endothelial cells and Erdogdu et al. obtained similar results with exendin-4 (GLP-1 receptor agonist) using human coronary artery endothelial cells [15,16]. Here we demonstrate that the topical administration of GLP-1 prevents the RNA/DNA and proteins damage induced by RNS and ROS in an experimental model of DR. To the best of our knowledge this is the first study showing these beneficial e ffect of GLP-1 in the diabetic retina. It should be noted that systemic administration of GLP-1 analogs, by reducing blood glucose levels, may led to similar results in terms of oxidative stress, but our study provides evidence that these e ffects are directly mediated by GLP-1 and cannot be attributed to an improvement of blood glucose levels. In this regard, it should be emphasized that the topical (eye drops) administration of GLP-1 does not alter the blood glucose levels and that, therefore, the e ffects cannot be attributed to an improvement in these levels.

In the present study, we evaluated the protein levels in the retina of some antioxidant enzymes such as glutathione peroxidase, glutathione reductase, CuZnSOD and MnSOD after topical treatment with GLP-1. We found that glutathione peroxidase and glutathione reductase were higher in diabetic mice treated with GLP-1 eye drops, but without reach the statistical significance. This result agree with those obtained by Fernández-Millán et al. who found that GLP-1 was able to enhance the activity of both enzymes in beta cells (rat INS-1E cells) [17]. Regarding CuZnSOD and MnSOD, significantly higher levels in db/db mice treated with GLP-1 eye drops in comparison with vehicle were observed. Therefore, the topical administration of GLP-1 was able to prevent the diabetes-induced downregulation of CuZnSOD and MnSOD in the retina. Overall, our findings point to the enhancement of all these antioxidant enzymes as a significant mechanism of action of GLP-1.

In order to investigate the potential role of GLP-1 in the process of DNA repair we measured the Babam2 protein, which is encoded by the *Babam2* gene, also named *Bre.* The *Babam2* gene forms the Brca1-a complex in the nucleus of multiple cell types where its function consists in repairing DNA double strands breaks. Babam2 enables the Brca1-a complex to reach DNA damage sites. Shi et al. demonstrated that the deletion of Babam2 in fibroblasts leads to the accumulation of unrepaired DNA damage [11]. In the present study, we provide first evidence that GLP-1 increases the Babam2 protein, thus suggesting that DNA repair is another pleiotropic action of GLP-1. Co-labeling with the specific neuronal marker NeuN points to the ganglion cells as the main neuronal source of the Babam2 protein. In addition, we have confirmed our previous observation that GLP-1 upregulates Ki67 (an excellent marker of cellular proliferation), which also colocalized with NeuN. Taken together these findings sugges<sup>t</sup> that the antioxidant properties of GLP-1 are linked to its capacity to promote neurogenesis.
