Topical Administration of GLP-1 Receptor Agonists Prevents Retinal Neurodegeneration in Experimental Diabetes

Systemic liraglutide administration. A: Glial activation; comparison of GFAP immunoreactivity (green) in the retina among representative samples from diabetic mice treated with vehicle, liraglutide, and restrictive diet and from a nondiabetic mouse. Nuclei were labeled with Hoechst stain (blue). B: Quantification of glial activation based on the extent of GFAP staining. The scoring system was as follows: Müller cell endfeet region/GCL only (score 1); Müller cell endfeet region/GCL plus a few proximal processes (score 2); Müller cell endfeet plus many processes, but not extending to ONL (score 3); Müller cell endfeet plus processes throughout with some in the ONL (score 4); and Müller cell endfeet plus lots of dark processes from GCL to outer margin of ONL (score 5). C: Apoptosis; TUNEL-positive immunofluorescence (green) in a representative mouse from each group. D: Percentage of TUNEL-positive cells in the neuroretina. Results are the mean ± SD. n = 10 mice per group. One-way ANOVA and Bonferroni multiple comparison test were used. *P < 0.05 compared with the other groups. C, control mice; D, diabetic mice.

A: Immunofluorescence showing the increase of GLP-1 (magenta) in the retina after liraglutide administration in representative samples from a diabetic mouse treated with subcutaneous liraglutide and a diabetic mouse treated with eye drops containing liraglutide. A representative sample from a diabetic mouse treated with vehicle (sham) and a nondiabetic mouse are also shown. Nuclei were labeled with Hoechst stain (blue). Arrows show how the liraglutide reached the retina. B: Quantification of GLP-1 immunofluorescence. n = 4 mice (8 eyes) per group. Results are the mean ± SD. One-way ANOVA and the Bonferroni multiple comparison test were used. C: Immunofluorescence staining for GLP-1 (magenta) in sections of neuroretina (middle panels) and ciliary body from a diabetic mouse showing a dose-dependent liraglutide accumulation. Mice were treated with a single dose of liraglutide at different concentrations (Dose 1 80 μg [n = 4], Dose 2 180 μg [n = 4], Dose 3 240 μg [n = 4]). After 60 min of topical administration, the mice were killed and the retinas were processed. C, control mice; D, diabetic mice.

Topical administration of GLP-1R agonists. A: Comparison of GFAP immunoreactivity (green) in the retina between representative samples from diabetic mice treated with vehicle and GLP-1R agonists (native GLP-1, lixisenatide, liraglutide, and exenatide) and a nondiabetic mouse. Nuclei were labeled with Hoechst stain (blue). B: Quantification of glial activation based on the extent of GFAP staining. C: Apoptosis; TUNEL-positive immunofluorescence (green) in a representative mouse from each group. D: Percentage of TUNEL-positive cells in the neuroretina. n = 6 mice per group. Results are the mean ± SD. One-way ANOVA and Bonferroni multiple comparison test were used. *P < 0.05 compared with the other groups. C, control mice; D, diabetic mice.

A: ERG traces in response to stimulus intensity of 40 cd/s/m² in a representative nondiabetic mouse (dark blue), a db/db mouse treated with vehicle (red), a db/db mouse treated with liraglutide (lira) by the subcutaneous route (gray), and a db/db mouse treated by the topical ocular route (green). cd, candela. B: Prevention of the decrease in ERG a-wave amplitude by liraglutide (subcutaneously and topically administered). C: Prevention of the decrease in ERG b-wave amplitude by liraglutide (subcutaneously and topically administered). D: Liraglutide (subcutaneously and topically administered) prevented the increase of OPs implicit time. We added up OP implicit time (ΣOP implicit time) for the first five OPs. Black columns, at day 14; white columns, baseline. n = 10 mice/group. Results are the mean ± SD. Differences between final and baseline ERG parameters in each group were assessed by paired Student t test. *P < 0.05. D, diabetic mice.

A: Comparison of cAMP immunofluorescence (green) among representative retinal samples from a db/db mouse treated with sham, a db/db mouse treated with liraglutide (lira) eye drops, and a nondiabetic mouse. Nuclei were labeled with Hoechst stain (blue). B: Detailed images of GCL showing the expression of cAMP (white arrows). C: Quantification of cAMP immunofluorescence in arbitrary units (A.U.). n = 10 mice per group. Results are the mean ± SD. One-way ANOVA and Bonferroni multiple comparison test were used. D: Representative Western blot analysis and quantification of the expression of pAKT and AKT in the neuroretina. Tubulin was used as a loading control. Immunoreactive pAKT was normalized to the total AKT, and the quotient of controls was set to unity. n = 6 mice per group. *P < 0.001 compared with the diabetic group treated with vehicle (D-Sham). C, control mice; D, diabetic mice.

A: Retinal concentration of glutamate measured by high-performance liquid chromatography in the following groups: db/db mice treated with vehicle (black bars), db/db mice treated subcutaneously with liraglutide (lira) (chessboard bars), db/db mice treated with liraglutide eye drops (vertical stripes bars), and nondiabetic mice (white bars). B: Comparison of GLAST immunofluorescence (red) among representative samples from a db/db mouse treated with vehicle, a db/db mouse treated with liraglutide subcutaneously, a db/db mouse treated with liraglutide eye drops, and a nondiabetic mouse. Nuclei were labeled with Hoechst stain (blue). C: Quantification of GLAST immunofluorescence in arbitrary units (A.U.). n = 10 mice per group. Results are the mean ± SD. One-way ANOVA and Bonferroni multiple comparison tests were used. *P < 0.001 compared with the other groups. C, control mice; D, diabetic mice.