Attenuation of Retinal Vascular Development in Neonatal Mice Subjected to Hypoxic-Ischemic Encephalopathy

Abstract

A significant proportion of children that survive hypoxic-ischemic encephalopathy (HIE) develop visual impairment. These visual deficits are generally attributed to injuries that occur in the primary visual cortex and other visual processing systems. Recent studies suggested that neuronal damage might also occur in the retina. An important structure affecting the viability of retinal neurons is the vasculature. However, the effects of HIE on the retinal neurovasculature have not been systemically evaluated. Here we investigated whether exposure of postnatal day 9 (P9) neonatal mice to HIE is sufficient to induce neurovascular damage in the retina. We demonstrate that the blood vessels on the surface of the retina, from mice subjected to HIE, were abnormally enlarged with signs of degeneration. The intermediate and deep vascular layers in these retinas failed to form normally, particularly in the periphery. All the vascular damages observed here were irreversible in nature up to 100 days post HIE. We also observed loss of retinal neurons, together with changes in both astrocytes and Müller cells mainly in the inner retina at the periphery. Collectively, our findings suggest that HIE results in profound alterations in the retinal vasculature, indicating the importance of developing therapeutic strategies to protect neurovascular dysfunction not only in the brain but also in the retina for infants exposed to HIE.

Figure 1

Hypoxic-ischemic insult disrupts the vascular development in the murine neonatal retina. Representative photomicrographs were taken from the middle to the periphery of cryosectioned P12 control and P9D3 HIE retinas. Blood vessels were labeled with Collagen IV and all retinal nuclei were labeled with DAPI. Collagen IV staining (red) demonstrate an approximate complete coverage of the deep vascular layer (arrow) to the periphery of the P12 control retinas (arrow head) but not of the P9D3 HIE retinas. Eyes from three mice per group were examined. Scale bar, 20 µm.

Figure 2

Hypoxic-ischemic insult disrupts the vascular development in the murine neonatal retina. Representative photomicrographs from the middle to the periphery of the retinas were taken from P9 control, P12 control, P9D3 HIE, P100 control, and P9D90 HIE mice. Blood vessels were labeled with Collagen IV or Griffonia simplicifolia isolectin B4. Vessels in different retinal layers were given pseudo-colors (Red, deep vascular layer; Green, intermediate vascular layer; blue, superficial layer). Because of retinal thinning, the superficial vascular layer at the edge of periphery is at the same plane of the lagging deep vascular layer; Both layers are separated by the white solid line. Retinas from at least five mice per group were examined. Scale bar, 50 µm.

Figure 3

Hypoxic-ischemic insult disrupts the vascular development in the murine neonatal retina as shown by trypsin digest preparations. Representative photomicrographs were taken from the peripheral retinal vasculature of P110 control, P9D100 mildly injured HIE and P9D100 severely injured HIE retinas. The nuclei of endothelial cells lie within the vessel wall along the axis of the capillary (empty arrow), while pericyte nuclei generally have a protuberant position on the capillary wall (solid arrow). Retinas from four control, 3 mild and 6 severe HIE mice were examined (numbers shown in the bars). The bars show the estimated mean cell counts/ratios. The error bars are 95% confidence intervals. Both means and CIs were estimated using the linear mixed effects models. * Indicates that by our criteria the HIE severe group is significantly different from control group. Images were captured on Aperio slide scanner and x20 objective was used.

Figure 4

Hypoxic-ischemic insult occasionally results in the formation of neovascularization tufts. Representative photomicrographs were taken from P12 control, P9D3 HIE, and P9D30 HIE retinas (A), P7H5N1, P7H5N3, and P7H5N27 retinas and (B), P9H5N1, P9H5N3, and P9H5N25 retinas (C). Blood vessels were labeled with Griffonia simplicifolia isolectin B4 or collagen IV. Neovascular tufts are marked by arrows. Arrowheads indicate unique abnormal vascular structures that do not protrude toward the vitreous. Retinas from at least four mice per time-point were examined. Scale bar, 200 µm.

Figure 5

Astrocyte distribution and density were altered in the retinas of HI mice. Representative photomicrographs were taken from P12 and P100 control and P9D3 and P9D90 HIE mice. Flat-mount retinas were labeled for isolectin B4 and GFAP, an intermediate filament protein expressed in astrocytes an in activated Müller cells. Two separate maximum projection images were generated per representative sample at the superficial and deep vascular layers. Retinas from control animals showed typical astrocyte distribution and density (stellate-shaped astrocytes which makes discrete contacts on blood vessels). On the other hand, the majority of HI retinas showed altered astrocytes distribution and density and activation of Müller cells. Arrows represent the activated Müller cells. Retinas from at least five mice per group were examined. Scale bar, 200 µm.

Figure 6

Hypoxic-ischemic insult causes degeneration of murine neonatal neuroretina. Eyes were processed and paraffin-sectioned through the optic disc then hematoxylin and eosin stained. Representative photomicrographs were taken for the whole eye section and from the center, the middle and the periphery of the eye. Postnatal day 9 (P9) control eye represents the eye structure before the hypoxia-ischemia was initiated; P12 control eye; P9D3 HIE represent P12 mouse (3 days post-HI); P100 control eye; P9D90 represent P99 mouse (90 days post-HI). Abbreviations: GCL: ganglion cell layer; IPL: inner plexiform layer; INL: inner nuclear layer; OPL: outer plexiform layer; and ONL: outer nuclear layer. Images were captured on Aperio slide scanner. Eyes from three mice per group were examined. Scale bar, 200 µm.