Increased neuronal nitric oxide synthase activity in retinal neurons in early diabetic retinopathy

Abstract

Purpose: There are increased levels of nitric oxide (NO) in diabetic retinas. The purpose of this study was to determine the extent that neuronal nitric oxide synthase (nNOS) contributes to the increased levels of retinal NO in early diabetic retinopathy by examining the expression and activity of nNOS in retinal neurons after 5 weeks of diabetes.

Methods: Changes in NO levels were measured using NO imaging of retinal neurons in mice with streptozotocin-induced diabetes for five weeks. NO imaging was compared to nNOS localization using immunocytochemistry, and nNOS message and protein levels were measured using quantitative real-time PCR and western blots.

Results: There was a close anatomic correlation between the localization of the increased NO production and the nNOS immunoreactivity in the retinal plexiform layers of diabetic retinas. There was no change in nNOS message, but nNOS protein was decreased and its subcellular localization was altered. Treatment with insulin or aminoguanidine partially ameliorated the increase in NO in diabetic retinas.

Conclusions: These results suggest that increased nNOS activity is responsible for the majority of increased NO in retinal neurons in early diabetic retinopathy. This supports a role for increased nNOS activity in the early neuronal dysfunction in the diabetic retina.

Figure 1

Changes in NO-induced fluorescence (NO-IF) in the diabetic retina. A: NO-IF increased in the diabetic retina compared to control. NO-IF in processes from somata in the inner nuclear layer (INL) were present in control (white arrowhead) but absent in the diabetic. Treatment with insulin or aminoguanidine (AG) reduced NO-IF and insulin restored NO-IF in some the processes in the INL (white arrowheads). Scale bars represent 25 μm. Quantitative analysis of NO-IF in retinal regions is shown in dark-adapted unstimulated (B) and light-stimulated retinas (C) as compared to control (100%). The outer and inner plexiform layers (OPL and IPL) depict the percent intensity, while the inner nuclear layer (INL) and ganglion cell layer (GCL) depict the percent of NO-IF labeled somata. B, C: Like symbols indicate statistically significant differences between groups (* represents diabetic versus control OPL, # represents diabetes versus control IPL; p<0.05). Error bars represent SD.

Figure 2

nNOS protein levels decreased after 5 weeks of diabetes. A: Mean fluorescence levels of nNOS immunoreactivity were significantly reduced in the outerplexiform layer (OPL), inner nuclear layer (INL), inner plexiform layer (IPL), and ganglion cell layer (GCL) when compared to controls (n=11). The graph on the right indicates the line profile average of the intensity of nNOS in each layer of the retina. B: nNOS immunoreactivity in the IPL no longer filled neuronal processes in the diabetic and was localized in structures which resembled synaptic boutons in size and location. C: western blots detected a single ~160 kDa band consistent with nNOS. Levels of β-tubulin are shown to confirm equal protein loading. There was a 24%±11 decrease in total nNOS protein (C is control, D is diabetic; n=8). Line on the left indicates the location of the 150 kDa molecular weight marker.

Figure 3

qPCR measurements of retinal nNOS transcripts using an Applied Biosystems TaqMan™ assay. No difference was seen between control and diabetic nNOS mRNA levels. The values are depicted as mean normalized expression (MNE) compared to the 18s rRNA (n=4). The data were analyzed using the qGene template [30], using the following equation: $\begin{array}{l}\text{MNE}=[\frac{{(E_{ref})}^{C{T}_{ref}},replicate1}{{(E_{target})}^{C{T}_{target}},replicate1}+\frac{{(E_{ref})}^{C{T}_{ref}},replicate2}{{(E_{target})}^{C{T}_{target}},replicate2}+\frac{{(E_{ref})}^{C{T}_{ref}},replicate3}{{(E_{target})}^{C{T}_{target}},replicate3}+\frac{{(E_{ref})}^{C{T}_{ref}},replicate4}{{(E_{target})}^{C{T}_{target}},replicate4}]\\ \\ 4\end{array}$where; E_target, PCR amplification efficiency of the target gene; E_ref, PCR amplification efficiency of the reference gene; CT_target, threshold cycle of the PCR amplification of the target gene; CT_ref, threshold cycle of the PCR amplification of the reference gene. The CT was defined as the cycle at which the fluorescence rises appreciably above the background fluorescence and determined automatically using Applied Biosystems SDS software version 2.3.