*2.2. Interventional Study*

When the mice reached the age of 21 weeks, GLP-1 eye drops (*n* = 15) and vehicle (phosphate-buffered saline (PBS) eye drops (*n* = 15) were randomly dispensed directly onto the superior corneal surface of both eyes with the help of a micropipette. They received one drop in each eye (5 μL) twice daily for 21 days. On the last day of treatment, at the age of 24 weeks, a drop of GLP-1 (2 mg/mL) or vehicle was administered to each eye 1 h before euthanasia. This study obtained the approval of the Animal Care and Use Committee of VHIR (Vall d'Hebron Research Institute, Barcelona, Spain). All experiments were performed in accordance with the guidelines of the European Community (86/609/CEE) and the Association for Research in Vision and Ophthalmology (ARVO).

#### *2.3. Retinal Tissue Processing*

On the last day of the topical administrations, 8 db/db mice and 4 db/+ were transcardially perfused with paraformaldehyde 4% (Santa Cruz Biotechnology, Dallas, TX, USA), and the eyes were promptly enucleated, fixed again in paraformaldehyde 4% for 5 h and embedded in paraffin blocks. Previously, each animal had received an intraperitoneal injection of 200 μL of anesthesia (a mix containing 1 mL ketamine (GmbH, Hameln, Germany) and 0.3 mL xylazine (Laboratorios Calier S.A., Barcelona, Spain)). The remaining mice (22 db/db and 11 db/+ mice) were euthanized through cervical dislocation, their eyes were instantaneously enucleated, and retinas were separated depending on the experimental purposes. For experiments that required protein samples, retinas were introduced in sterilized PBS pH 7.4 and frozen in nitrogen liquid. For RNA assessments retinas were submerged in TRIzol reagen<sup>t</sup> (InvitrogenTM, Carlsbad, CA, USA) and stored at −80 ◦C until analysis.
