Topical administration of somatostatin prevents retinal neurodegeneration in experimental diabetes

Abstract

Retinal neurodegeneration is an early event in the pathogenesis of diabetic retinopathy (DR). Somatostatin (SST) is an endogenous neuroprotective peptide that is downregulated in the diabetic eye. The aim of the study was to test the usefulness of topical administration of SST in preventing retinal neurodegeneration. For this purpose, rats with streptozotocin-induced diabetes mellitus (STZ-DM) were treated with either SST eye drops or vehicle for 15 days. Nondiabetic rats treated with vehicle served as a control group. Functional abnormalities were assessed by electroretinography (ERG), and neurodegeneration was assessed by measuring glial activation and the apoptotic rate. In addition, proapoptotic (FasL, Bid, and activation of caspase-8 and caspase-3) and survival signaling pathways (BclxL) were examined. Intraretinal concentrations of glutamate and its main transporter glutamate/aspartate transporter (GLAST) were also determined. Treatment with SST eye drops prevented ERG abnormalities, glial activation, apoptosis, and the misbalance between proapoptotic and survival signaling detected in STZ-DM rats. In addition, SST eye drops inhibited glutamate accumulation in the retina and GLAST downregulation induced by diabetes mellitus. We conclude that topical administration of SST has a potent effect in preventing retinal neurodegeneration induced by diabetes mellitus. In addition, our findings open up a new preventive pharmacological strategy targeted to early stages of DR.

FIG. 1.

Ocular absorption of ¹²⁵I-somatostatin expressed as the percentage of applied dose for each gram of tissue (percent dose/g) as a function of time. Uptake of the radioactivity in the eye content was clearly observed (n = 1 for each time point).

FIG. 2.

The b-wave amplitude (A) and implicit time (B) under scotopic conditions (0 db) in both eyes at day −1 (□) and at day 14 (■) in the studied groups. C, control rats; D-sham, diabetic rats treated with vehicle eye drops; D-SST, diabetic rats treated with SST eye drops. Data are means ± SD. *P < 0.05.

FIG. 3.

GFAP immunofluorescence. A: Images of representative samples of retina from a control rat (Control), a diabetic rat treated with vehicle eye drops (D-Sham), and a diabetic rat treated with SST eye drops (D-SST). In diabetic rats, the endfeet of the Müller cells showed abundant GFAP immunofluorescence (green), and the radial processed stained intensely throughout the inner retina. The topical treatment with SST prevents the glial activation. Scale bar = 50 μm. B: Quantification of glial activation based on extent of GFAP staining. INL, inner nuclear layer.

FIG. 4.

A: Representative images of apoptosis in the retina from a control rat, a diabetic rat treated with vehicle eye drops (Diabetes-sham), and a diabetic rat treated with SST eye drops (Diabetes-SST). B: Percentage of apoptotic cells in the whole retina and in the retinal layers in each group. Results are expressed as means ± SD. INL, inner nuclear layer. *P < 0.05 in comparison with control and diabetic SST treatment groups. Scale bar = 50 μm.

FIG. 5.

Dose-response effect of SST eye drops on retinal neurodegeneration. A: GFAP immunofluorescence (green) corresponding to representative samples of retina from a control rat (A1), a diabetic rat treated with vehicle eye drops (A2), and diabetic rats treated with SST eye drops at doses of 0.5 mg/mL (A3), 2 mg/mL (A4), and 10 mg/mL (A5). B: Quantification of glial activation based on extent of GFAP staining corresponding to all rats (n = 8) of each group. C: Images of apoptosis assessed by TUNEL assay (green) in representative samples of retina from a control rat (C1), a diabetic rat treated with vehicle eye drops (C2), and diabetic rats treated with SST eye drops at doses of 0.5 mg/mL (C3), 2 mg/mL (C4), and 10 mg/mL (C5). D: Percentage of apoptotic cells in the retinal layers in each group (n = 8). Results are expressed as median (range). Control, control rats; D-sham, diabetic rats treated with vehicle eye drops; D-SST, diabetic rats treated with SST eye drops; INL, inner nuclear layer. *P < 0.05 in comparison with diabetic group treated with vehicle. Scale bar = 20 μm.

FIG. 6.

Apoptotic signaling pathways in the neuroretina of three groups. A: Protein extracts were prepared from neuroretina. Total protein (50 μg) was used for Western blot analysis with the antibodies against FasL, caspase (casp)-8, Bid, Bim, BclxL, and active caspase-3. α-Tubulin (αtub) antibody was used as a loading control. B: Autoradiograms were quantified by scanning densitometry. C, control rats; D-Sham, diabetic rats treated with vehicle eye drops; D-SST, diabetic rats treated with SST eye drops. Results are expressed as arbitrary units of protein expression and are means ± SD. *P < 0.05, **P < 0.01, and ***P < 0.005 for diabetic mice treated with the vehicle vs. diabetic mice treated with SST eye drops.

FIG. 7.

A: GLAST mRNA in control rats (C), diabetic rats treated with vehicle eye drops (D-Sham), and diabetic rats treated with SST eye drops (D-SST). B: Immunofluorescence of GLAST in representative samples of retina from each group. C: Quantification of GLAST immunofluorescence in the three studied groups. Data are means ± SD. a.u., arbitrary units; INL, inner nuclear layer. Scale bar = 50 μm.