Combination Antioxidant/NSAID Therapies and Oral/Topical Ocular Delivery Modes for Prevention of Oxygen-Induced Retinopathy in a Rat Model

Abstract

Given the complexity of oxygen-induced retinopathy (OIR), we tested the hypothesis that combination therapies and modes of administration would synergistically optimize efficacy for prevention of OIR. Newborn rats were exposed to neonatal intermittent hypoxia (IH) from the first day of life (P0) until P14 during which they received: (1) oral glutathione nanoparticles (nGSH) with topical ocular phosphate buffered saline (PBS); (2) nGSH with topical ocular Acuvail (ACV); (3) oral coenzyme Q10 (CoQ10) + ACV; (4) oral omega 3 polyunsaturated fatty acids (n-3 PUFAs) + ACV; (5) CoQ10 + n-3 PUFAs + PBS; or (6) CoQ10 + n-3 PUFAs + ACV. Treated groups raised in room air (RA) served as controls. At P14, pups were placed in RA with no treatment until P21. Retinal vascular pathology, ocular angiogenesis biomarkers, histopathology, and morphometry were determined. All combination treatments in IH resulted in the most beneficial retinal outcomes consistent with suppression of angiogenesis growth factors during reoxygenation/reperfusion and no significant adverse effects on somatic growth. nGSH + PBS also reversed IH-induced retinopathy, but had negative effects on growth. Simultaneously targeting oxidants, inflammation, and poor growth mitigates the damaging effects of neonatal IH on the developing retina. Therapeutic synergy with combination delivery methods enhance individual attributes and simultaneously target multiple pathways involved in complex diseases such as OIR.

Keywords: coenzyme Q10; glutathione nanoparticles; insulin-like Growth Factor-I; ketorolac; n-3 polyunsaturated fatty acids; neonatal intermittent hypoxia; oxygen-induced retinopathy; vascular endothelial growth factor.

Figure 1

Expression of HIF_1α in the retina and choroid in response to combination treatments in RA and IH by Western blot analysis. Data are presented as mean ± SEM ratio of HIF_1α/GAPDH (n = 4 replicates). Comparison among the RA or IH groups was conducted using one-way ANOVA with Bonferroni post hoc multiple comparisons test. Comparison between RA and IH for each group was conducted using unpaired t-test. Retinal HIF_1α/GAPDH ratios (A), Choroidal HIF_1α/GAPDH ratios (B).

Figure 2

Effects of combination treatments on vascular endothelial growth factor (VEGF) levels in the serum (A), vitreous fluid (B), retina (C), and choroid (D). Levels in the retinal and choroidal homogenates were standardized using total cellular protein levels. The open bar represents the room air (RA) groups and the solid bar represents the IH groups. Data are expressed as mean ±SD (n = 6 samples/group for serum; and n = 4 samples/group for vitreous fluid, retina and choroid).

Figure 3

Effects of combination treatments on soluble vascular endothelial growth factor receptor (sVEGFR)-1 levels in the serum (A), vitreous fluid (B), retina (C), and choroid (D). Levels in the retinal and choroidal homogenates were standardized using total cellular protein levels. The open bar represents the room air (RA) groups and the solid bar represents the IH groups. Data are expressed as mean ±SD (n = 6 samples/group for serum; and n = 4 samples/group for vitreous fluid, retina and choroid).

Figure 4

Effects of combination treatments on soluble vascular endothelial growth factor receptor (sVEGFR)-2 levels in the serum (A), retina (B), and choroid (C). sVEGFR-2 was not detected in the vitreous fluid. Levels in the retinal and choroidal homogenates were standardized using total cellular protein levels. The open bar represents the room air (RA) groups and the solid bar represents the IH groups. Data are expressed as mean ±SD (n = 6 samples/group for serum; and n = 4 samples/group for vitreous fluid, retina and choroid).

Figure 5

Effects of combination treatments on insulin-like growth factor (IGF)-I levels in the serum (A), vitreous fluid (B), retina (C), and choroid (D). Levels in the retinal and choroidal homogenates were standardized using total cellular protein levels. The open bar represents the room air (RA) groups and the solid bar represents the IH groups. Data are expressed as mean ±SD (n = 6 samples/group for serum; and n = 4 samples/group for vitreous fluid, retina, and choroid).

Figure 6

Representative H&E stained corneas from neonatal rats on postnatal day 21 (P21). Room air (RA) groups are represented in the top panels and intermittent hypoxia (IH) groups are represented in the bottom panels. Images are 40× magnification, scale bar is 20 µm.

Figure 7

Representative ADPase-stained retinas from room air (RA) raised neonatal rats at postnatal day 21 (P21). The upper panels represent the periphery and the lower panels represent the optic disk. Images are 10× magnification, scale bar is 100 µm.

Figure 8

Representative ADPase-stained retinas from rats exposed to neonatal intermittent hypoxia (IH) at postnatal day 21 (P21). The upper panels represent the periphery and the lower panels represent the optic disk. Images are 10× magnification, scale bar is 100 µm. Arrow heads show retinal damage.

Figure 9

Representative glial fibrillary acidic protein (GFAP) (green)/isolectin B (red) stained merged images of retinas from room air (RA) raised neonatal rats at postnatal day 21 (P21). The upper panels represent the periphery and the lower panels represent the optic disk. Images are 10× magnification, scale bar is 100 µm.

Figure 10

Representative GFAP (green)/isolectin B (red) stained merged images of retinas from rats exposed to neonatal intermittent hypoxia (IH) at postnatal day 21 (P21). The upper panels represent the periphery and the lower panels represent the optic disk. Images are 10× magnification, scale bar is 100 µm.

Figure 11

Representative H&E stained retinal layers from neonatal rats on postnatal day 21 (P21). Room air (RA) groups are represented in the top panels and intermittent hypoxia (IH) groups are represented in the bottom panels. Images are 40× magnification, scale bar is 20 µM.