Intravitreal Administration of AAV2-SIRT1 Reverses Diabetic Retinopathy in a Mouse Model of Type 2 Diabetes

Abstract

Purpose: The expression of silent information regulator (SIRT) 1 is reduced in diabetic retinopathy (DR). Previous studies showed that alterations in SIRT1 messenger RNA (mRNA) and protein expression are implicated in progressive inflammation and formation of retinal acellular capillaries. Treatment with the SIRT1 agonist, SRT1720, improved visual response by restoration of a- and b-wave responses on electroretinogram scotopic measurements in diabetic (db/db) mice. In this study, we investigated the effects of intravitreal SIRT1 delivery on diabetic retinal pathology.

Methods: Nine-month-old db/db mice received one intravitreal injection of either AAV2-SIRT1 or AAV2-GFP control virus, and after 3 months, electroretinography and optomotor responses were measured. Their eyes were then removed and analyzed by immunohistochemistry and flow cytometry.

Results: SIRT1 mRNA and protein levels were increased following AAV2-SIRT1 administration compared to control virus AAV2-GFP injected mice. IBA1+ and caspase 3 expression were decreased in retinas of db/db mice injected with AAV2-SIRT1, and reductions in scotopic a- and b-waves and high spatial frequency in optokinetic response were prevented. Retinal hypoxia inducible factor 1α (HIF-1α) protein levels were reduced in the AAV2-SIRT1-injected mice compared to control-injected mice. Using flow cytometry to assess changes in intracellular HIF-1α levels, endothelial cells (CD31+) from AAV-2 SIRT1 injected mice demonstrated reduced HIF-1α expression compared to db/db mice injected with the control virus.

Conclusions: Intravitreal AAV2-SIRT1 delivery increased retina SIRT1 and transduced neural and endothelial cells, thus reversing functional damage and improving overall visual function.

Translational relevance: AAV2-SIRT1 gene therapy represents a beneficial approach for the treatment of chronic retinal conditions such as DR.

Figure 1.

AAV2-SIRT1 intravitreal injection increases SIRT1 expression in retina cells. (A) The retina of mice injected with AAV2-SIRT1 shows increased SIRT1 immunohistochemical staining compared with diabetic retina. (B) SIRT1 mRNA expression is increased in the retina of AAV2-SIRT1–treated mice compared to diabetic mice with control virus treatment. (C) AAV2-SIRT1 increases the number of SIRT1 immunopositive cells.

Figure 2.

AAV2-SIRT1 treatment decreases retinal inflammation. (A) IBA1⁺ cells in the retina of control mice, diabetic mice, and diabetic mice following AAV2-SIRT1 injection. (B) Quantification of IBA1⁺ cells in each experimental cohort. AAV2-SIRT1 injection decreases the number of IBA1⁺ cells in the retina. (C) Inflammatory cells are significantly reduced in the retina of AAV2-SIRT1–treated diabetic mice compared to control virus–treated mice.

Figure 3.

AAV2-SIRT1 treatment decreases retinal reactive gliosis. (A) GFAP staining in the retina of control mice, diabetic mice, and diabetic mice with the AAV2-SIRT1 treatment. (B) Quantification of GFAP intensity in each of the different experimental groups. AAV2-SIRT1 injection decreases the intensity of GFAP staining in the retina of treated diabetic mice.

Figure 4.

AAV2-SIRT1 treatment reduces the number of acellular capillaries and apoptosis. (A) Representative images of retinal acellular capillary staining in control, diabetic, and diabetic mice injected with AAV2-SIRT1. Red arrows point to acellular capillaries. (B) Quantification of acellular capillaries. Number of capillaries is significantly increased in the diabetic retina compared to the control retina and is decreased in the retina of the diabetic mice injected with AAV-2 SIRT. (C) Immunohistochemistry for caspase 3 in retinal sections. White arrows point to caspase 3⁺ cells. (D) Quantification of caspase 3⁺ cells in the retina. The number of apoptotic caspase 3⁺ cells is reduced in the diabetic mice injected with AAV2-SIRT1 compared to control virus–injected mice.

Figure 5.

SIRT1 overexpression reduces retinal endothelial cell hypoxia. (A) Flow cytometry gating strategy for total HIF-1α in the retina. (B) Gating strategy for CD31⁺ endothelial cells. The first column represents the gating for the percentage of CD31⁺ cells in the retina. The second column shows the percentage of the CD31⁺ cells that are HIF-1α⁺. (C) Percentage of HIF-1α⁺ cells in the total retina, CD31⁺ cells, bipolar cells (PKC-α⁺), and rod photoreceptors (rhodopsin⁺ cells).

Figure 6.

AAV2-SIRT1 improves visual recovery. (A) ERG scotopic a-wave amplitudes for mice at light intensities of −20, −10, and 0 db. Intravitreal AAV2-SIRT1 injection improves the amplitude of the scotopic a-wave at −10 db compared to diabetic mice; however, at 0 db, the amplitudes are similar for both diabetes and diabetes with AAV2-SIRT1 treatment. (B) Isolated ERG response amplitudes for the −10 db light intensity. (C) Optomotor response measurements. The spatial frequency observed by the diabetic mice is significantly reduced compared to the control mice and diabetic mice injected with intravitreal AAV2-SIRT1.

Figure 7.

AAV2 injection does not cause systemic inflammation. (A) Percentage of classical and nonclassical monocytes in the blood. There were no significant differences in the percentage of classical and nonclassical monocytes in blood of diabetic mice compared to controls. (B) Percentage of classical and nonclassical monocytes over total number of monocytes. There were no significant changes in the percentage of classical and nonclassical monocytes in the bone marrow of control mice with and without treatment and diabetic mice with and without AAV2 treatment. The AAV2 injections did not alter the percentages of the classical and nonclassical monocytes in the bone marrow of the diabetic mice.