Hyperoxia-Induced Proliferative Retinopathy: Early Interruption of Retinal Vascular Development with Severe and Irreversible Neurovascular Disruption

Abstract

Bronchopulmonary dysplasia (BPD) is a major cause of neonatal morbidity in premature infants, occurring as a result of arrested lung development combined with multiple postnatal insults. Infants with BPD exposed to supplemental oxygen are at risk of retinopathy of prematurity as well. Thus, we studied the effects of hyperoxia on the retinal vasculature in a murine model of BPD. The retinal phenotype of this model, which we termed hyperoxia-induced proliferative retinopathy (HIPR), shows severe disruption of retinal vasculature and loss of vascular patterning, disorganized intra-retinal angiogenesis, inflammation and retinal detachment. Neonatal mice were subjected to 75% oxygen exposure from postnatal day (P)0 to P14 to model BPD, then allowed to recover in room air for 1 (P15), 7 (P21), or 14 days (P28). We quantified retinal thickness, protein levels of HIF-1α, NOX2, and VEGF, and examined the cellular locations of these proteins by immunohistochemistry. We examined the retinal blood vessel integrity and inflammatory markers, including macrophages (F4/80) and lymphocytes (CD45R). Compared to controls, normal retinal vascular development was severely disrupted and replaced by a disorganized sheet of intra-retinal angiogenesis in the HIPR mice. At all time-points, HIPR showed persistent hyaloidal vasculature and a significantly thinner central retina compared to controls. HIF-1α protein levels were increased at P15, while VEGF levels continued to increase until P21. Intra-retinal fibrinogen was observed at P21 followed by sub-retinal deposition in at P28. Inflammatory lymphocytes and macrophages were observed at P21 and P28, respectively. This model presents a severe phenotype of disrupted retinal vascular development, intra-retinal angiogenesis inflammation and retinal detachment.

Fig 1. Disorganized angiogenesis in hyperoxia-induced proliferative retinopathy.

(A) Representative flat mounts from P14, P21, and P28 mice in control mice (RA) and HIPR were stained with IB4 (N = 3–4). Scale bar, 500 μm. (B) Left image is an inset of the red box of P21 HIPR in (A). Right image is an inset of the green box. Persistent hyaloidal vasculature is present and attached to the retina in the mid-periphery. Scale bar, 300 μm. (C) Retinal cross sections stained with IB4 (red), GFAP (green), and DAPI (blue) of HIPR retinas show a persistent hyaloidal vessel (arrow) close to the retinal surface (N = 3–4). Scale bar, 50 μm. RGCL, retinal ganglion cell layer.

Fig 2. Elevated HIF-1α levels in hyperoxia-induced proliferative retinopathy.

(A) Retinal HIF-1α was evaluated by western blotting, quantified by densitometry, and normalized to β-actin. Representative blots shown. HIF-1α levels significantly increased from P14 to P15 and then decreased by P21. Data were fold change ± SEM (n ≥ 4 per group). (B) Retinal cross sections were immunostained with HIF-1α (black, N = 3) and counterstained with methyl green. HIF-1α was found in the retinal ganglion cell layer and in the nuclei of the inner nuclear layer (asterisk). Scale bar, 100 μm. ONL, outer nuclear layer. INL, inner nuclear layer. RGCL, retinal ganglion cell layer. IPL, inner plexiform layer.

Fig 3. VEGF is localized to retinal ganglion cell layer and inner plexiform layer in hyperoxia- induced proliferative retinopathy retinas.

(A) Retinal protein VEGF levels were quantified with an ELISA. VEGF increased until P21 and then decreased. Values were mean ± SEM (N = 4). # p<0.05 HIPR P15 compared to HIPR P14. * p<0.05 HIPR to respective age-matched RA controls. § p<0.05 HIPR P21 compared to HIPR P15. (B) Sections were immunostained with VEGF (red) and co-stained with an endothelial cell marker, CD31 (green), and DAP1 (blue, N = 2). VEGF was found in the RGCL at P15 and mainly in IPL at P21. Persistent hyaloidal vessels stained strongly for VEGF and CD31 and these hyaloidal vessels were in close proximity to the inner retina (arrow). INL, inner nuclear layer. RGCL, retinal ganglion cell layer. IPL, inner plexiform layer. Scale bar, 50 μm.

Fig 4. Retinal VEGFR2 levels increased in hyperoxia-induced proliferative retinopathy.

Immunohistochemistry revealed VEGFR2 (red) cellular locations. Sections were stained with GFAP (green) for activated Mϋller cells (N = 3). In HIPR, VEGFR2 was co-localized with GFAP by P21. High intensity VEGFR2 staining was seen in the persistent hyaloidal vessels in HIPR. INL, inner nuclear layer. RGCL, retinal ganglion cell layer. IPL, inner plexiform layer. Scale bar, 50 μm.

Fig 5. Increased NOX2 expression in hyperoxia-induced proliferative retinopathy.

(A, B) Retinal NOX2 was evaluated by western blotting, quantified by densitometry, and normalized to β-actin. (A) Representative western blots are shown. The vertical black line indicates separate samples were run between these lanes on the same blot. (B) Quantification of westerns show that NOX2 levels significantly increased from P14 to P15 and from P15 to P21. Data were fold change ± SEM (n ≥ 5 per group). * p<0.05 HIPR to respective age-matched RA controls. § p<0.05 HIPR P21 compared to HIPR P15. (C) Retinal cross sections were immunostained with NOX2 (green), IB4 (red), and counterstained with DAPI (blue, N = 3). In RA, NOX2 was only seen in blood vessels. In HIPR, NOX2 was observed in the retinal ganglion cell layer. Scale bar, 50 μm. INL, inner nuclear layer. IPL, inner plexiform layer. RGCL, retinal ganglion cell layer.

Fig 6. Retinal thinning near the optic nerve and in the peripheral retina in hyperoxia-induced proliferative retinopathy.

(A) Cross sections of P21 RA (top) and HIPR (bottom) showing persistent hyaloidal vessels (blue asterisk). Insets (blue rectangles) correspond to higher magnification images in (B). Scale bar, 500 μm. L, lens. V, vitreous. (B) Cross sections were H&E stained (N = 3–4). In HIPR at P21 and P28, the outer nuclear layer and inner nuclear were not distinguishable, indicating severe thinning of the outer plexiform layer. Scale bar, 50 μM. ONL, outer nuclear layer. INL, inner nuclear layer. RGCL, retinal ganglion cell layer. PR, photoreceptors. The thickness of retinal layers at 100 μm from the optic nerve (C) or in the retinal periphery (D). (C) Near the optic nerve, HIPR had thinner retinas at all time points compared to RA. (D) At P15, P21, and P28, the peripheral HIPR retinas were significantly thinner than RA. * p<0.05 HIPR to respective age-matched, RA. § p<0.05 HIPR P21 compared to HIPR P15. Values were mean ± SEM (N = 3–4).

Fig 7. Fibrinogen present in the inner retina in hyperoxia-induced proliferative retinopathy.

(A) Retinal cross sections were immunostained with fibrinogen (green), IB4 (red), and counterstained with DAPI (blue, N = 3). No fibrinogen staining was seen in the retina or the vitreous of RA mice. However, fibrinogen staining was visible in the inner nuclear layer, inner plexiform layer (arrow), and ganglion cells in HIPR. Fibrinogen staining was also observed in the vitreous of HIPR at P21 and P28, and in the sub-retinal space at P28 HIPR. Arrowhead indicates persistent hyaloidal vasculature. Scale bar, 50 μm. INL, inner nuclear layer. IPL, inner plexiform layer. RGCL, retinal ganglion cell layer. RPE, retinal pigment epithelium.

Fig 8. White blood cells present in the inner plexiform layer in hyperoxia-induced proliferative retinopathy.

(A) Retinal cross sections were immunostained with (A) F4/80 (red) or (B) CD45 (green) and counterstained with DAPI (blue, N = 3). In RA, there was no F4/80 or CD45 staining. In HIPR, F4/80 and CD45 were both seen in the IPL at P21 and P28. Scale bar, 50 μm. INL, inner nuclear layer. IPL, inner plexiform layer. RGCL, retinal ganglion cell layer.

Fig 9. Collagen fibers found in hyperoxia-induced proliferative retinopathy.

Retinal cross sections were stained with Masson’s trichrome (N = 3). Collagen fibers were detected in P21 and P28 HIPR retinas (arrows). Red blood cells were seen in the vitreous in the P28 HIPR with a nearby persistent hyaloidal vessel (arrowhead). Scale bar, 100 μm. RGCL, retinal ganglion cell layer. V, vitreous.

Fig 10. Schematic of downstream events in hyperoxia-induced proliferative retinopathy model.

Upon removal from 75% oxygen at P14, there are no retinal vessels seen except for the persistent hyaloidal vessels (blue). By P15, the surge in HIF-1α leads to an increase in VEGF and angiogenesis (red) in the central retina and mid-periphery. At P21, there is disorganized angiogenesis (red) in the IPL and increased VEGF and NOX2. Fibrinogen and CD45+ lymphocytes are observed in the retina. By P28, the retina is thin and collagen fibers were detected in the vitreous, indicating traction retinal detachment. Fibrinogen (green) was found in the vitreous and sub-retinal space (suggestive of exudative detachment) and hemorrhages were seen in the vitreous (red dots). CD45+ lymphocytes and F4/80+ macrophages are increased at P28.