Diabetes Renders Photoreceptors Susceptible to Retinal Ischemia-Reperfusion Injury

Abstract

Purpose: Studies have suggested that photoreceptors (PR) are altered by diabetes, contributing to diabetic retinopathy (DR) pathology. Here, we explored the effect of diabetes on retinal ischemic injury.

Methods: Retinal ischemia-reperfusion (IR) injury was caused by elevation of intraocular pressure in 10-week-old BKS db/db type 2 diabetes mellitus (T2DM) mice or C57BL/6J mice at 4 or 12 weeks after streptozotocin (STZ)-induced type 1 diabetes mellitus (T1DM), and respective nondiabetic controls. Retinal neurodegeneration was evaluated by retinal layer thinning, TUNEL staining, and neuron loss. Vascular permeability was evaluated as retinal accumulation of circulating fluorescent albumin. The effects of pretreatment with a sodium-glucose co-transporter (SGLT1/2) inhibitor, phlorizin, were examined.

Results: Nondiabetic control mice exhibited no significant outer retinal layer thinning or PR loss after IR injury. In contrast, db/db mice exhibited significant outer retina thinning (49%, P < 0.0001), loss of PR nuclei (45%, P < 0.05) and inner segment (IS) length decline (45%, P < 0.0001). STZ-induced diabetic mice at 4 weeks showed progressive thinning of the outer retina (55%, by 14 days, P < 0.0001) and 4.3-fold greater number of TUNEL+ cells in the outer nuclear layer (ONL) than injured retinas of control mice (P < 0.0001). After 12 weeks of diabetes, the retinas exhibited similar outer layer thinning and PR loss after IR. Diabetes also delayed restoration of the blood-retinal barrier after IR injury. Phlorizin reduced outer retinal layer thinning from 49% to 3% (P < 0.0001).

Conclusions: Diabetes caused PR to become highly susceptible to IR injury. The ability of phlorizin pretreatment to block outer retinal thinning after IR suggests that the effects of diabetes on PR are readily reversible.

Figure 1.

Retinal layer thinning after IR injury in type 2 diabetic mice. At 10 weeks of age (2–6 weeks of diabetes), db/+ (control), and db/db (diabetic) mice were subjected to retinal IR injury. OCT (A) was used to evaluate total (B), inner (C), and outer (D) retinal thickness at 10 days following IR injury (n = 5–8/group). At 14 days following IR injury, retinas were plastic-embedded, sectioned, and stained with Lee's stain (E) to evaluate total (F), inner (G), and outer (H) retinal thickness. The linear density of nuclei in the ONL (I) and the length of IS (J) were also evaluated. Significances of differences between groups: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001, with colors indicating the comparison of db/db IR with each of the other groups.

Figure 2.

Time course of retinal layer thinning after IR injury in STZ-induced diabetic mice. After 4 weeks of STZ-induced diabetes, the mice were subjected to retinal IR injury. OCT (A) was used to evaluate total (B), inner (C), and outer (D) retinal thickness before IR injury and at the indicated times after IR injury (n = 8–12/group). Significances of differences between groups: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001, with colors indicating the comparison of STZ-IR group with each other group.

Figure 3.

Retinal layer thinning after IR injury in STZ-induced diabetic mice with longer duration of diabetes. After 3 months of STZ-induced diabetes, the mice were subjected to retinal IR injury. OCT (A) was used to evaluate total (B), inner (C), and outer (D) retinal thickness at 10 days following IR injury (n = 5–8/group). At 14 days following IR injury, retinas were plastic-embedded, sectioned, and stained with Lee's stain (E) to evaluate the linear density of nuclei in the ONL (F) and the length of IS (G). Significances of differences between groups: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001, with color indicating the comparison of STZ-IR with each of the other groups.

Figure 4.

TUNEL staining in retinal sections in STZ-induced diabetic mice after IR injury. After 4 weeks of STZ-induced diabetes, the mice were subjected to retinal IR injury. At 1 and 4 days after injury, the retinas (n = 5/group) were harvested, embedded, thin sectioned, and TUNEL stained (A). TUNEL positive cells in different retinal layers were quantified at 1 day (B) and 4 days (C) after IR injury. Significances of differences between groups: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001.

Figure 5.

TUNEL staining in flat-mount retinas in STZ-induced diabetic mice after IR injury. After 4 weeks of STZ-induced diabetes, mice were subjected to retinal IR injury. At 2 and 4 weeks after injury, the retinas (n = 5/group) were harvested, flat-mounted, and stained for TUNEL, IB4 (vascular), and Iba-1 (microglia and invading phagocytes). Representative images from OPL and ONL at 2 weeks after IR injury are shown (A). TUNEL positive cells in different retinal layers were quantified at 2 weeks (B) and 4 weeks (C) after IR injury. Retinal cell death assays were performed at 2 and 4 weeks after IR injury (D), with optical density of the DNA fragmentation ELISA normalized to mg of retina wet weight in each reaction. Significances of differences between groups: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001.

Figure 6.

Effect of IR injury on retinal permeability in STZ-induced diabetic mice. At 4 weeks after STZ-induced diabetes, mice were subjected to IR injury. At the indicated times following IR injury, the retinal permeability was assessed with FITC-BSA (A). At 4 weeks after STZ-induced diabetes, the mice were subjected to IR injury. At 4 weeks following IR injury, the extravascular accumulation of sulfo-NHS-biotin was imaged to visualize vascular leakage (B). Intensity of NHS-biotin in the flat-mount retinas were quantified (C). Confocal images show NHS-biotin in the deep plexus (D). Significances of differences between groups: *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001.

Figure 7.

Effect of a SGLT1/2 inhibitor on retinal layer thinning. After 4 weeks of STZ-induced diabetes, the mice were treated twice (16 and 1.5 hours before ischemia) with an SGLT inhibitor (phlorizin, 400 mg/kg, intraperitoneally [IP]) or carrier prior to retinal IR injury. OCT (A) was used to evaluate total (B), inner (C), and outer (D) retinal thickness at 10 days following IR injury (n = 8/group). At 14 days following IR injury, retinas were plastic-embedded, sectioned and stained with Lee's stain (E) to evaluate total (F), inner (G), and outer (H) retinal thickness. The linear density of nuclei in the ONL (I) and the length of IS (J) were also evaluated. Significances of differences between groups: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001, with red color in F to J indicating the comparison between the STZ-vehicle-IR and the STZ-phlorizin-IR groups.