Ocular Adverse Effects of Intravitreal Bevacizumab Are Potentiated by Intermittent Hypoxia in a Rat Model of Oxygen-Induced Retinopathy

Abstract

Intravitreal bevacizumab (Avastin) use in preterm infants with retinopathy of prematurity is associated with severe neurological disabilities, suggesting vascular leakage. We examined the hypothesis that intermittent hypoxia (IH) potentiates intravitreal Avastin leakage. Neonatal rats at birth were exposed to IH from birth (P0)-P14. At P14, the time of eye opening in rats, a single dose of Avastin (0.125 mg) was injected intravitreally into the left eye. Animals were placed in room air (RA) until P23 or P45 for recovery (IHR). Hyperoxia-exposed and RA littermates served as oxygen controls, and equivalent volume saline served as the placebo controls. At P23 and P45 ocular angiogenesis, retinal pathology and ocular and systemic biomarkers of angiogenesis were examined. Retinal flatmounts showed poor peripheral vascularization in Avastin-treated and fellow eyes at P23, with numerous punctate hemorrhages and dilated, tortuous vessels with anastomoses at P45 in the rats exposed to IH. These adverse effects were associated with robust increases in systemic VEGF and in both treated and untreated fellow eyes. Histological analysis showed severe damage in the inner plexiform and inner nuclear layers. Exposure of IH/IHR-induced injured retinal microvasculature to anti-VEGF substances can result in vascular leakage and adverse effects in the developing neonate.

Figure 1

Effects of Avastin on retinal (a, b), vitreous fluid (c, d), and choroidal (e, f) VEGF levels in adolescent 23-day-old (a, c, and e) and pubertal 45-day-old (b, d, and f) rats exposed to room air (RA), hyperoxia (50% O₂), and intermittent hypoxia (IH). Animals exposed to IH received 50% O₂ with brief, clustered hypoxia (12% O₂) episodes for a total of 8 episodes per day from P0 to P14. Data are presented as mean ± SD (n = 6 samples/group). ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001 versus saline RA; ^†p < 0.05, ^‡p < 0.01 versus Avastin RA; ^§p < 0.05, ^§§p < 0.01, ^§§§p < 0.001 versus right untreated eye. RE: right eye; LE: left eye.

Figure 2

Effects of Avastin on retinal (a, b), vitreous fluid (c, d), and choroidal (e, f) sVEGFR-1 levels in adolescent 23-day-old (a, c, and e) and pubertal 45-day-old (b, d, and f) rats exposed to room air (RA), hyperoxia (50% O₂), and intermittent hypoxia (IH). Groups are as described in Figure 1. Data are presented as mean ± SD (n = 6 samples/group). ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001 versus saline RA; ^†p < 0.05, ^‡p < 0.01 versus Avastin RA; ^§p < 0.05, ^§§p < 0.01, versus right untreated eye. RE: right eye; LE: left eye.

Figure 3

Effects of Avastin on retinal (a, b), vitreous fluid (c, d), and choroidal (e, f) IGF-1 levels in adolescent 23-day-old (a, c, and e) and pubertal 45-day-old (b, d, and f) rats exposed to room air (RA), hyperoxia (50% O₂), and intermittent hypoxia (IH). Groups are as described in Figure 1. Data are presented as mean ± SD (n = 6 samples/group). ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001 versus saline RA; ^†p < 0.05, ^‡p < 0.01 versus Avastin RA; ^§p < 0.05, ^§§p < 0.01 versus right untreated eye. RE: right eye; LE: left eye.

Figure 4

Retinal flatmounts showing ADPase-stained retinas from 23-day-old rat exposed to RA (a–d), 50% O2 (e–h), and IH (i–l). (a), (e), and (i) are right untreated retinas from saline-treated groups; (b), (f), and (j) are right untreated retinas from Avastin-treated groups; (c), (g), and (k) are saline-treated left retinas; and (d), (h), and (l) are Avastin-treated left retinas. (a) is a representative image of the WimRetina analysis for quantitation of vascular density parameters. Images are 10x magnification. Scale bar, 100 μm.

Figure 5

Retinal flatmounts showing ADPase-stained retinas from 45-day-old rats. Groups are as described in Figure 4. Images are 10x magnification. Scale bar, 100 μm.

Figure 6

H&E stain of retinal layers from 23-day-old rat exposed to RA (a–d), 50% O₂ (e–h), and IH (i–l). (a), (e), and (i) are right untreated retinas from saline-treated groups; (b), (f), and (j) are right untreated retinas from Avastin-treated groups; (c), (g), and (k) are saline-treated left retinas; and (d), (h), and (l) are Avastin-treated left retinas. Images are 40x magnification. Scale bar, 20 μm.

Figure 7

H&E stain of retinal layers from 45-day-old rats. Groups are as described in Figure 6. Images are 40x magnification. Scale bar, 20 μm.