Retinal protective effects of resveratrol via modulation of nitric oxide synthase on oxygen-induced retinopathy

Abstract

Purpose: Retinopathy of prematurity (ROP) is one of the leading causes of blindness, with retinal detachment occurring due to oxygen toxicity in preterm infants. Recently, advances in neonatal care have led to improved survival rates for preterm infants, and ROP has increased in incidence. In the present study, we aimed to determine whether or not resveratrol exhibits protective effects in an animal model of ROP and in primary retinal cell cultures of neonatal rat via nitric oxide (NO)-modulating actions using western blotting and real-time PCR with inducible nitric oxide synthase (iNOS), endothelial NOS (eNOS) and neuronal NOS (nNOS) antibodies and mRNAs.

Methods: In an in vivo oxygen-induced retinopathy (OIR) model, cyclic hyperoxia was induced with 80% O(2) for one day and 21% O(2) for one day from P1 to P14 in newborn Sprague-Dawley (SD) rats. Resveratrol was injected intravitreally for seven days and rats were sacrificed at P21. In vitro OIR primary retinal cell culture was performed using P0-2 SD rats. Hyperoxia injuries were induced through 100% O(2) exposure for six hours. Western blotting and real-time PCR using iNOS, eNOS, nNOS antibodies and primers were performed in the rat model of ROP and the dispersed retinal cell culture.

Results: In both in vivo and in vitro OIR, the expression of iNOS antibody and mRNA was increased and of eNOS and nNOS were reduced in the resveratrol-treated group.

Conclusions: In conclusion, resveratrol appeared to exert retinal protective effects via modulation of NO-mediated mechanism in in vivo and in vitro OIR models.

Keywords: Nitric oxide synthase; Resveratrol; Retinopathy of prematurity.

Fig. 1

Morphologic analysis after H&E staining revealed changes in the major retinal layers of 21-day-old Sprague-Dawley rats. (A) The N group did not show any retinal pathology. (B) In striking contrast, the H group presented retinal lesions displaying retinal neovascularization, fibrovascular growth, and large areas with blood vessel leakage like retinal detachment. (C) The HR group shows a few holes which are not retinal detachment. N=normoxia; H=hyperoxia; HR=hyperoxia+resveratrol; OSL=outer segment layer; ISL=inner segment layer; ONL=outer nuclear layer; OPL=outer plexiform layer; INL=inner nuclear layer; IPL=inner plexiform layer; GCL=ganglion cell layer; ILM=inner limiting membrane.

Fig. 2

(A)Western blots for inducible nitric oxide synthase (iNOS), endothelia nitric oxide synthase (eNOS), and neuronal nitric oxide synthase (nNOS) in the in vivo oxygen-induced retinopathy (OIR) were revealed. Relative expressions of iNOS (B), eNOS (C), and nNOS (D) in the N, H, and HR groups were revealed. The HR group was administered with 30 mg/kg of resveratrol before a hyperoxic insult. N=normoxia; H=hyperoxia; HR=hyperoxia+resveratrol. ^*p<0.05, statistically significant.

Fig. 3

Real-time PCR for inducible nitric oxide synthase (iNOS), endothelia nitric oxide synthase (eNOS), and neuronal nitric oxide synthase (nNOS) in the in vivo oxygen-induced retinopathy (OIR) were revealed. Relative expressions of iNOS (A), eNOS (B), and nNOS (C) in the N, H, and HR groups were revealed. The HR group was administered with 30 mg/kg of resveratrol before a hyperoxic insult. N=normoxia; H=hyperoxia; HR=hyperoxia+resveratrol. ^*p<0.05, statistically significant.

Fig. 4

Resveratrol attenuation in the in vivo oxygen-induced retinopathy (OIR). Cultured dispersed retinal cells were prepared with different concentrations of resveratrol for 30 min before hyperoxic insult for 6 hours. Cell viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. The concentrations of the drugs were 5, 10, 50 and 100 ug/mL. N=normoxia; H=hyperoxia; HR=hyperoxia+resveratrol. ^*p<0.05, statistically significant vs. N.

Fig. 5

(A) Immunofluorescence of photoreceptor marker IRBP (interphotoreceptor retinoid-binding protein) using a fluorescence microscope under high magnification (normoxia group, ×200), and photomicrographs of cultured dispersed retinal cells with a phase contrast microscope (×100). The N (B), H (C), and HR (D) groups were revealed. N=normoxia; H=hyperoxia; HR=hyperoxia+resveratrol.

Fig. 6

(A) Western blots for inducible nitric oxide synthase (iNOS), endothelia nitric oxide synthase (eNOS), and neuronal nitric oxide synthase (nNOS) in the in vitro oxygen-induced retinopathy (OIR) were revealed. Relative expressions of iNOS (B), eNOS (C), and nNOS (D) in the N, H, and HR groups were revealed. The HR group was administered with 5 ug/mL of resveratrol before a hyperoxic insult. N=normoxia; H=hyperoxia; HR=hyperoxia+resveratrol. ^*p<0.05, statistically significant.

Fig. 7

Real-time PCR for inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS), and neuronal nitric oxide synthase (nNOS) in the in vitro oxygen-induced retinopathy (OIR) were revealed. Relative expressions of iNOS (A), eNOS (B), and nNOS (C) in the N, H, and HR groups were revealed. The HR group was administered with 5 ug/mL of resveratrol before a hyperoxic insult. N=normoxia; H=hyperoxia; HR=hyperoxia+resveratrol. ^*p<0.05, statistically significant.