Ocular Versus Oral Propranolol for Prevention and/or Treatment of Oxygen-Induced Retinopathy in a Rat Model

Abstract

Purpose: Propranolol, a nonselective B1/B2 adrenoceptor antagonist, promotes the regression of infantile hemangiomas likely through suppression of vascular endothelial growth factor (VEGF), which prompted its use for the prevention of retinopathy of prematurity. We tested the hypothesis that topical ocular propranolol is safe and effective for reducing the severity of oxygen-induced retinopathy (OIR) in the neonatal rat intermittent hypoxia (IH) model. Methods: At birth (P0), rat pups were randomly assigned to room air or neonatal intermittent hypoxia (IH) consisting of 50% O₂ with brief episodes of hypoxia (12% O₂) from P0 to P14, during which they received a single daily dose of oral propranolol (1 mg/kg/day in 50 μL in sterile normal saline) or topical ocular propranolol (0.2% in 10 μL in normal saline) from P5 to P14. Placebo-controlled littermates received 50 μL oral or 10 μL topical ocular sterile normal saline. Retinal vascular and astrocyte integrity; retinal histopathology and morphometry; and angiogenesis biomarkers were determined. Results: Topical ocular propranolol improved retinal vascular damage and preserved the astrocytic template, but did not completely prevent OIR. The beneficial effects of propranolol were associated with reduced ocular VEGF and increased endogenous soluble inhibitor, sVEGFR-1, when administered topically. Conclusions: Propranolol failed to completely prevent severe OIR, however, it prevented astrocyte degeneration resulting from neonatal IH-induced damage. We conclude that the mechanisms of propranolol's beneficial effects in neonatal IH may involve in part, astrocyte preservation.

Keywords: angiogenesis; hypoxia; neovascularization; propranolol; retina.

FIG. 1.

Representative ADPase-stained retinas from rats raised in RA at postnatal day 21 (P21) and treated with topical ocular or oral saline or propranolol. The upper panels are peripheral vessels, and the lower panels are vessels at the optic disk. Pups were administered a single daily treatment of topical ocular or oral propranolol or saline occurred from P5 to P14. Topical ocular propranolol caused hemorhrage (arrow) and oral propranolol in caused tortuous vessels (arrow). Images are 10 × magnification, scale bar is 100 μm. ADPase, adenosine diphosphatase; RA, room air. Color images are available online.

FIG. 2.

Representative ADPase-stained retinas from rats exposed to neonatal IH at postnatal day 21 (P21) and treated with topical ocular or oral saline or propranolol. The upper panels are peripheral vessels, and the lower panels are vessels at the optic disk. Pups were administered a single daily treatment of topical ocular or oral propranolol or saline occurred from P5 to P14. Saline treatment in neonatal IH resulted in hemorrhage, vessel tortuosity, vascular abundance, and neovascularization (arrows) which were not prevented with propranolol (arrows). Images are 10 × magnification, scale bar is 100 μm. IH, intermittent hypoxia. Color images are available online.

FIG. 3.

Representative GFAP (green)/isolectin B (red)-stained retinas from rats raised in RA at postnatal day 21 (P21) and treated with topical ocular or oral saline or propranolol. The upper panels are peripheral vessels, and the lower panels are vessels at the optic disk. Pups were administered a single daily treatment of topical ocular or oral propranolol or saline occurred from P5 to P14. Images are 10 × magnification, scale bar is 100 μm. GFAP, glial fibrillary acid protein. Color images are available online.

FIG. 4.

Representative GFAP (green)/isolectin B (red)-stained retinas from rats exposed to neonatal IH at postnatal day 21 (P21) and treated with topical ocular or oral saline or propranolol. The upper panels are peripheral vessels, and the lower panels are vessels at the optic disk. Pups were administered a single daily treatment of topical ocular or oral propranolol or saline occurred from P5 to P14. Saline treatment in neonatal IH caused astrocyte damage and gliosis (arrows) which was prevented with propranolol treatment. Images are 10 × magnification, scale bar is 100 μm. Color images are available online.

FIG. 5.

Representative H&E-stained retinas from rats raised in RA (upper panels) or neonatal IH (lower panels) at postnatal day 21 (P21) and treated with topical ocular or oral saline or propranolol. Pups were administered a single daily treatment of topical ocular or oral propranolol or saline occurred from P5 to P14. Saline treatment in neonatal IH increased NFL/GCL layer and number of endothelial cells, as did propranolol treatment in RA and neonatal IH (arrows). Images are 40 × magnification, scale bar is 20 μm. H&E, Hematoxylin and Eosin; INL, inner nuclear layer; IPL, inner plexiform layer; NFL/GCL, nerve fiber layer/ganglion cell layer; ONL, outer nuclear layer; R/C, rods and cones. Color images are available online.

FIG. 6.

Representative H&E-stained corneas from rats raised in RA (upper panels) or neonatal IH (lower panels) at postnatal day 21 (P21) and treated with topical ocular or oral saline or propranolol. Pups were administered a single daily treatment of topical ocular or oral propranolol or saline occurred from P5 to P14. Images are 40 × magnification, scale bar is 20 μm. Color images are available online.

FIG. 7.

Effects of topical ocular or oral propranolol or placebo saline on VEGF levels in the vitreous fluid at P14 (A) and P21 (B). The open bars represent the topical ocular administration and the solid bars represent oral administration. Data are expressed as mean ± SD (n = 4 samples/group). SD, standard deviation; VEGF, vascular endothelial growth factor.

FIG. 8.

Effects of topical ocular or oral propranolol or placebo saline on VEGF levels in the retina at P14 (A) and P21 (B) and in the choroid at P14 (C) and P21 (D). Levels were standardized using total cellular protein levels. The open bar represents the RA groups and the solid bar represents the IH groups. Data are expressed as mean ± SD (n = 6 samples/group).

FIG. 9.

Effects of topical ocular or oral propranolol or placebo saline on sVEGFR-1 levels in the retina at P14 (A) and P21 (B) and in the choroid at P14 (C) and P21 (D). Levels were standardized using total cellular protein levels. The open bar represents the RA groups and the solid bar represents the IH groups. Data are expressed as mean ± SD (n = 6 samples/group). sVEGFR, soluble vascular endothelial growth factor receptor.

FIG. 10.

Effects of topical ocular or oral propranolol or placebo saline on sVEGFR-2 levels in the retina at P14 (A) and P21 (B) and in the choroid at P14 (C) and P21 (D). Levels were standardized using total cellular protein levels. The open bar represents the RA groups and the solid bar represents the IH groups. Data are expressed as mean ± SD (n = 6 samples/group).

FIG. 11.

Effects of topical ocular or oral propranolol or placebo saline on IGF-I levels in the retina at P14 (A) and P21 (B) and in the choroid at P14 (C) and P21 (D). Levels were standardized using total cellular protein levels. The open bar represents the RA groups and the solid bar represents the IH groups. Data are expressed as mean ± SD (n = 4 samples/group). IGF, insulin-like growth factor.