Inhibition of inducible nitric oxide synthase reverses the loss of functional hyperemia in diabetic retinopathy

Abstract

Neuronal activity leads to arteriole dilation and increased blood flow in retinal vessels. This response, termed functional hyperemia, is diminished in the retinas of diabetic patients, possibly contributing to the development of diabetic retinopathy. The mechanism responsible for this loss is unknown. Here we show that light-evoked arteriole dilation was reduced by 58% in a streptozotocin-induced rat model of type 1 diabetes. Functional hyperemia is believed to be mediated by glial cells and we found that glial-evoked vasodilation was reduced by 60% in diabetic animals. The diabetic retinas showed neither a decrease in the thickness of the retinal layers nor an increase in neuronal loss, although signs of early glial reactivity and an upregulation of inducible nitric oxide synthase (iNOS) were detected. Inhibition of iNOS restored both light- and glial-evoked dilations to control levels. These findings suggest that high NO levels resulting from iNOS upregulation alters glial control of vessel diameter and may underlie the loss of functional hyperemia observed in diabetic retinopathy. Restoring functional hyperemia by iNOS inhibition may limit the progression of retinopathy in diabetic patients.

Fig. 1. Light-evoked vasodilation is reduced in diabetic retinas

A,B: IR-DIC images of the vitreal surface of the retina, illustrating the light-evoked responses of small arterioles. In a control retina (A), light stimulation evokes a large vasodilation (at 17 and 45 s after onset of the light stimulus). In a diabetic retina (B), light evokes a smaller dilation (at 21 s), followed by a constriction (at 27 s). The diameter of both control and diabetic vessels recover to baseline after light stimulation ends. Solid white lines indicate baseline vessel diameter; dashed lines indicate changed diameter. Scale bar, 10 µm. C,D: Light-evoked arteriole dilation in a normal (C) and a diabetic (D) retina. Light stimulation evokes a smaller dilation, followed by a constriction, in the diabetic retina.

Fig. 2. Few overt signs of retinopathy are seen in the diabetic retina

A: Mean thickness of retinal layers is not reduced in diabetic animals. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; PR, photoreceptors. B,C: Cell death was not increased in diabetic retinas. Very few TUNEL-positive cells (green/yellow profiles) were observed in both control (B) and diabetic (C) retinas. DAPI-labeled cell nuclei are shown in red. D–G: Immunostaining shows the expression of GFAP (green) in control (D) and three diabetic (E–G) retinas. A range of GFAP expression was observed in the vertically oriented Müller cells in diabetic retinas. Retinas from some animals were similar to controls (E), some showed a minor increase (F) while some showed substantial upregulation (G). GFAP-positive astrocytes, located beneath the GCL, are seen in both control and diabetic retinas. DAPI-labeled cell nuclei are shown in red. Scale bars, 20 µm.

Fig. 3. Glial-evoked vasodilation is reduced in diabetic retinas

A,B: IR-DIC images of a normal (A) and diabetic (B) retina showing small arterioles. Pseudocolor images showing glial Ca²⁺ increases are superimposed. In A and B, the top two images show time points before and 1 s after photolysis of caged Ca²⁺ in single glial cells (black dots). The last images show time points at which maximum vessel dilation was observed, with maximum ΔF/F Ca²⁺ projections overlaid. Although the glial Ca²⁺ increases are similar in control and diabetic retinas, the glial-evoked dilation is smaller in the diabetic retina. Scale bar, 10 µm. C,D: Ca²⁺ increases (upper traces) and arteriole diameters (lower traces) in a control (C) and a diabetic (D) retina. Photolysis of caged Ca²⁺ (black dots) produces similar glial Ca²⁺ increases in control and diabetic retinas. Yet, glial stimulation produces smaller arteriole dilations in the diabetic retina.

Fig. 4. iNOS expression is increased in diabetic retinas

Immunostaining shows the expression of iNOS, nNOS, and eNOS (green) in control (B,F,J) and diabetic (D,H,L) retinas. DAPI staining (red) shows cell nuclei in control (A,E,I) and diabetic (C,G,K) retinas. iNOS expression in the ONL, IPL and GCL of diabetic retinas (D) is raised compared to controls (B). nNOS (F,H) and eNOS (J,L) expression is unaltered in diabetic retinas. Scale bar, 20 µm.

Fig. 5. Inhibition of iNOS activity restores light- and glial-evoked vasodilation in diabetic retinas

A–C: Light-evoked vasomotor responses in a control retina (A), diabetic retina (B) and a diabetic retina treated with aminoguanidine (AG, 100 µM; C). D: The incidence of light-evoked arteriole dilations and constrictions. The incidence of vasoconstrictions is increased in diabetic retinas and is restored to control levels by 1400W (1 µM) and AG (100 µM), two inhibitors of iNOS. E: The magnitude of light-evoked arteriole dilations and constrictions. The magnitude of vasodilations is reduced while that of vasoconstrictions is increased in diabetic retinas. 1400W and AG restore these vasomotor responses to control levels. F–H: Photolysis-evoked glial Ca²⁺ increases (upper traces) and the resulting vascular responses (lower traces) in a control retina (F), diabetic retina (G) and a diabetic retina treated with AG (100 µM; H). Black dots indicate photolysis of caged Ca²⁺. I: The incidence of glial-evoked arteriole dilations and constrictions. J. The magnitude of glial-evoked vasodilations and constrictions. The magnitude of vasodilations is reduced and vasoconstrictions increased in diabetic retinas. Both are restored to control levels by AG. K: Photolysis-evoked peak glial Ca²⁺ responses (measured in processes surrounding vessels) are not different between control, diabetic and AG-treated diabetic retinas. *p < 0.02.