Korean J Ophthalmol.  2012 Dec;26(6):455-461. 10.3341/kjo.2012.26.6.455.

Triamcinolone Acetonide Prevents Enhancement of Hypoxia-induced Neuronal and Inducible Nitric Oxide Synthases in the Retinas of Rats with Oxygen-induced Retinopathy

• Kim SJ
• Chung IY
• Choi WS
• Kim YH
• Yoo JM

• KMID: 1499685
• DOI: http://doi.org/10.3341/kjo.2012.26.6.455

Abstract

PURPOSE
We investigated whether oxygen-induced retinopathy (OIR) results in changes in the protein expression of neuronal and inducible nitric oxide synthases (nNOS and iNOS, respectively) in rat model of OIR. In addition, we evaluated whether treatment of rats with triamcinolone acetonide (TA) prevents this response.
METHODS
To promote OIR, Sprague-Dawley rats were exposed to hyperoxia from postnatal day 2 (P2) to P14. They were then returned to normoxia after P15. TA was injected into the right vitreous of P15 rats, while saline was injected into the left vitreous. At P18 the expression of nNOS and iNOS was determined using Western blotting and immunostaining techniques in retinas obtained from control rats.
RESULTS
In P18 OIR rats, the abundance of nNOS and iNOS protein was significantly increased compared with controls. These increases were not observed in the retinas of rats treated with TA. The change in expression of nNOS and iNOS were specific to parvalbumin and glial fibrillary acidic protein-positive cells. Treatment with TA prevented the increased expression of nNOS and iNOS in all samples.
CONCLUSIONS
Hypoxia upregulates expression of nNOS and iNOS in OIR rat retinas, which is can be prevented by treatment with TA.

Keyword

Anoxia; Nitric oxide synthase type I; Nitric oxide synthase type II; Oxygen-induced retinopathy; Triamcinolone acetonide

MeSH Terms

Animals
Animals, Newborn
Anoxia/metabolism/pathology/*prevention & control
Blotting, Western
Disease Models, Animal
Female
Glucocorticoids/pharmacology
Immunohistochemistry
Neurons/metabolism
Nitric Oxide Synthase Type II/*biosynthesis
Oxygen/toxicity
Pregnancy
*Pregnancy, Animal
Rats
Rats, Sprague-Dawley
Retina/*metabolism/pathology
Retinal Diseases/chemically induced/pathology/*prevention & control
Triamcinolone Acetonide/*pharmacology
Glucocorticoids
Triamcinolone Acetonide
Oxygen
Nitric Oxide Synthase Type II

Figure

• 

  Fig. 1 (A) Western blot of retinal neuronal nitric oxide synthase (nNOS) protein in postnatal day 18 (P18) oxygen-induced retinopathy (OIR) rats versus control rats. A single band of approximately 155 kDa was detected in total retina protein extracts of P18 OIR rats and control rats. Levels of nNOS protein in the retina increased in saline treated P18 OIR rats versus saline treated control rats, and this increase was prevented by treatment with triamcinolone acetonide (TA). (B) Bar graphs representing retinal nNOS protein expression showing significant changes between saline treated P18 OIR rats and saline treated control rats (**p < 0.001). The increase in expression of nNOS protein is markedly decreased in P18 OIR treated with TA (†p < 0.05) when compared with saline treated controls. (C) Western blot for inducible nitric oxide synthase (iNOS) protein in the retinas of P18 OIR rats versus control rats. A single band of approximately 130 kDa was detected in total retina protein extracts of P18 OIR rats and control rats. Levels of retinal iNOS protein increased in saline treated P18 OIR rats versus saline treated control rats. This increase was blocked by treatment with TA. (D) Bar graphs representing retinal iNOS protein expression showing significant changes between saline treated P18 OIR rats and saline treated control rats (**p < 0.001). A significant reduction in retinal iNOS protein was observed for rats treated with TA (†p < 0.05). TAA = triamcinolone acetonide; CTL = control.

• 

  Fig. 2 Photomicrographs of retinal coronal sections showing neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) immunoreactivity. (A) nNOS-immunoreactive cells were detected in the ganglion cell layer (GCL, presented as small arrow) and inner nuclear layer (INL, presented as large arrow) in saline treated control rats. (B) nNOS immunoreactivity was increased in the GCL and INL in saline treated postnatal day 18 (P18) oxygen-induced retinopathy (OIR) rats. (C) Treatment with triamcinolone acetonide (TA) restored nNOS immunoreactivity in the GCL and INL to levels similar to that of saline treated control rats. Scale bar = 25 µm. (D) Yellow-signals (presented as white arrow) co-stained with parvalbumin (red) and nNOS (green) were observed in the INL of saline treated OIR rats in double immunofluorescent staining. (E) Weak iNOS immunoreactivity was detected in the nerve fiber layer (NFL) in saline treated control rats. (F) iNOS immunoreactivity was markedly increased in the NFL (presented as arrowhead) in saline treated P18 OIR rats. (G) Treatment with TA prevented the increase in iNOS immunoreactivity in the retinas of P18 OIR rats. (H) Yellow-signals (presented as white arrowhead) co-stained with iNOS (red) and GFAP (green) were observed in the NFL of saline treated OIR rats in double immunofluorescent staining.

• 

  Fig. 3 (A) Graphical representation of the changes in magnitude of staining for neuronal nitric oxide synthase (nNOS)-positive amacrine cells in postnatal day 18 (P18) oxygen-induced retinopathy (OIR) rats versus control rats. nNOS-positive amacrine cells in saline treated P18 OIR rats were increased versus saline treated control rats (**p < 0.001). Treatment with triamcinolone acetonide (TA) prevented an increase of nNOS-positive cells (†p < 0.05). (B) Changes in the magnitude of staining for inducible nitric oxide synthase (iNOS) immunoreactivity in the nerve fiber layer of saline treated P18 OIR rats versus control rats. iNOS immunoreactivity in saline treated P18 OIR rats was versus saline treated control rats (**p < 0.001). Treatment with TA prevented the increase of iNOS immunoreactivity (†p < 0.05). TAA = triamcinolone acetonide; CTL = control.

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