Dynamic changes of inducible nitric oxide synthase expression in rat's retina and its role on blood-retinal barrier injury after acute high intraocular pressure

Abstract

Aim: To clarify the role of inducible nitric oxide synthase (iNOS) in blood-retinal barrier (BRB) injury after acute high intraocular pressure (IOP) in rats.

Methods: Forty-two Sprague-Dawley (SD) rats were randomized into 7 groups [control (Cont), 3, 6, 12, 24, 48, and 72h, n=6]. Except Cont group, other groups' retina tissue was obtained at corresponding time points after a model of acute high IOP have been established in rats. The expression of iNOS and tight junction protein zonula occludens (ZO)-1 was detected by Western blotting. Evans blue (EB; 3% ) was injected into the great saphenous vein to detect the leakage of EB by spectrophotometer. Nine rats were divided into Cont, 6h, 12h groups, the expression of iNOS was localized by immunofluorescence. In order to verify the role of iNOS in the damage to BRB, thirty-six rats were randomly divided into 4 groups [Cont, Cont+inhibitor (Inh), 6h and 6h+Inh, n=9]. After treatment with the iNOS-specific inhibitor 1400W, the expression of iNOS and ZO-1 and the leakage of BRB were detected again.

Results: The immunofluorescence results showed that the expression of iNOS was observed in the Cont group and 6h group, but not in the 12h group. iNOS was mainly expressed in the retinal nerve fiber layer, ganglion cell layer and inner nuclear layer and that it did not colocalize with the retinal ganglion cell marker NeuN but was co-expressed with the vascular endothelial cell marker CD31. Western blotting showed that in the early period (3h, 6h) after acute high IOP, the expression of iNOS was upregulated, then the down-regulation of iNOS were tested in the follow-up timing spots. ZO-1 expression showed a continuous down-regulation after 6h. The quantitative results for EB showed that the amount of EB leakage began to increase at 3h after acute high IOP. At 6h, the leakage of EB was lower, but at 12h, the leakage of EB was highest, after which it gradually recovered but remained higher than that in the Cont group. The expression of iNOS was down-regulated after 1400W treatment. ZO-1 expression was not significantly changed in the Cont+Inh group and the 6h group, and significantly down-regulated in the 6h+Inh group, and the leakage of EB was significantly increased after 1400W treatment.

Conclusion: These results suggest that the upregulation of iNOS expression in the early stage after acute high IOP may have a protective effect on BRB injury.

Keywords: ZO-1; acute high intraocular pressure; blood-retinal barrier; inducible nitric oxide synthase.

Figure 1. Double-label immunofluorescence staining of frozen retinal sections of rats

A: Double immunofluorescence staining of iNOS (red) and CD31 (green) in the retina of Cont group, 6h group and 12h group, with DAPI (blue) marking the nucleus. Bar=100 µm. B: Double-label immunofluorescence staining of iNOS (red) and NeuN (green) in the retina of the 6h group. The images on the next line were enlarged pictures of the box, and DAPI (blue) marks the nucleus. Bar=50 µm. NFL: Nerve fiber layer; GCL: Ganglion cell layer; IPL: Inner plexiform layer; INL: Inner nuclear layer; OPL: Outer plexiform layer; ONL: Outer nuclear layer; iNOS: Inducible nitric oxide synthase; CD31: Platelet endothelial cell adhesion molecule-1; DAPI: 4′,6-diamidino-2-phenylindole; NeuN: Neuron-specific nuclear protein.

Figure 2. Western blot detection of iNOS and ZO-1 expression changes at different time points after acute high IOP

A: The expression of iNOS and ZO-1 at 3h, 6h, 12h, 1d, 2d, and 3d after acute high IOP in rats; B, C: The statistical analysis diagrams of the expression levels of iNOS and ZO-1, respectively. ^aP<0.05 compared with the Cont group. iNOS: Inducible nitric oxide synthase; ZO: Zonula occludens; IOP: Intraocular pressure; Cont: Control.

Figure 3. EB quantitatively detects the amount of EB permeation at different time points after acute high IOP in rats

The longitudinal ordinate indicates the EB content, and the horizontal ordinate indicates the different time points. ^aP<0.05 compared with the Cont group. EB: Evans blue; IOP: Intraocular pressure; Cont: Control.

Figure 4. Western blot detection of the expression of iNOS and ZO-1 before and after inhibitor treatment in the Cont group and in the 6h group

A: The expression of iNOS and ZO-1 in the Cont group and the 6h and 6h+Inh groups after acute high IOP in rats; B, C: The statistical analysis diagrams of the expression levels of iNOS and ZO-1, respectively. ^aP<0.05 compared with the Cont group. iNOS: Inducible nitric oxide synthase; ZO: Zonula occludens; IOP: Intraocular pressure; Cont: Control; Inh: Inhibitor.

Figure 5. EB leakage of the Cont group and 6h group before and after inhibitor

A: Fluorescence image of retinal EB for the qualitative detection of BRB damage (Bar=200 µm); B: The amount of BRB leakage quantitatively detected by EB. ^bP<0.001 compared with the Cont group. EB: Evans blue; BRB: Blood-retinal barrier; Cont: Control; Inh: Inhibitor.