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Zeinab Nasralah, Ronald Hobbs, Faizah N Bhatti, Alistair J Barber, Penn State Hershey Eye Center; Nrf2 protein content is reduced after extended dark adaptation in retinas of diabetic rats. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1035.
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Nuclear factor E2-related factor-2 (Nrf2) is a transcription factor that responds to inflammatory signaling and intracellular superoxide to initiate the antioxidant response pathway. In this project, Nrf2 protein content was measured in retinas of diabetic and control rats in both light-adapted and dark-adapted conditions. Toll-like receptor 4 (TLR) mediated inflammatory signaling was used as a positive control.
Long-Evans rats were made diabetic by injection with streptozotocin (STZ, 65 mg/kg, pH 4.5, i.v., n=16) and compared to age-matched controls (CNT, n=16), housed for 4 or 10 weeks before sacrifice. For each experiment, half the STZ-diabetic and CNT rats were sacrificed in normal light-adapted conditions, while the other half were dark adapted for 24 hr and sacrificed in the dark. Nrf2 protein content in retina lysates was determined by immunoblot. As a positive control experiment C57Bl/6J mice and Long-Evans rats (n=4) were given intravitreal injections of bacterial lipopolysaccharide (LPS, 1 µg and 5 µg respectively) into one eye, and Nrf2 content was compared to the opposing saline-injected eye 6-24 hr later. Statistical comparisons were made by ANOVA with Newman-Keuls multiple comparisons test and p<0.05 considered a significant difference.
Nrf2 content was unchanged after 4 and 10 weeks in STZ-diabetic rats compared to CNT when sacrificed in the light. Nrf2 content was significantly lower in dark-adapted STZ-diabetic rats compared to light-adapted groups after 10 weeks of hyperglycemia, but not 4 weeks (p<0.05). In contrast, Nrf2 protein was elevated in mouse retinas 12 hr after intraocular injection of LPS, and peaked after 18 hr, remaining high after 24 hr. A similar significant increase in Nrf2 protein content occurred 18 hr after intraocular LPS injection in rats (p<0.015).
Nrf2 protein content was not altered in diabetic rats sacrificed in the light, but was significantly reduced in dark-adapted diabetic rats. This suggests that dark adaptation transiently down regulates retinal Nrf2 protein in diabetic rats. In contrast Nrf2 was dramatically elevated in the retina after TLR-4 mediated inflammatory signaling. We postulate that Nrf2 down regulation during dark adaptation in diabetes is by a mechanism that does not involve TLR signaling.
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