In retinopathy of prematurity (ROP), elevated levels of oxygen arrest the normal posterior-to-anterior growth of intraretinal blood vessels; in some patients, the demarcation zone between vascular and avascular retina thickens into an ophthalmoscopically visible ridge that designates stage 2 ROP. The ridge consists of a larger anterior collection of spindle-shaped cells in the nerve fiber layer (the “vanguard”) and a variably present smaller posterior vascularized rearguard.1 Although the ridge is an important aspect of ROP pathogenesis, its cellular composition has not been well characterized.
Four eyes were obtained at autopsy from 2 children clinically diagnosed with stage 2 ROP at the Stanford University Medical Center.
A male baby was born at 24 weeks' gestational age and treated with oxygen for respiratory distress from birth. Stage 2 ROP was diagnosed 8 weeks after birth. The child died at age 12 weeks of multiple organ failure.
A male baby was born at 27 weeks with hydrops fetalis and an 11p13-15.1 deletion. The patient received supplemental oxygen and died 6 weeks after birth.
Immunohistochemical examination was performed on formalin-fixed paraffin-embedded sections using antisera to identify mature and immature vascular endothelial cells (CD31; Dako, Glostrup, Denmark, or CD34; Becton, Dickinson, and Co, Franklin Lakes, New Jersey), astrocytes (glial fibrillary acidic protein [GFAP]; Dako), astrocyte precursor cells (PAX2; Zymed Laboratories, San Francisco, California), myeloid lineage cells (microglia and macrophages; CD68; Dako), pericytes (desmin; Dako), neurons (neuron-specific enolase; Dako), or proliferating cells (Ki67; Dako).
A thickened ridge of spindle cells (the vanguard) in the nerve fiber layer just anterior to the most distal blood vessels was verified in eosin-stained sections in the temporal and nasal aspects of all 4 eyes; a smaller vascular proliferation in the rearguard was evident in some cases (Figure 1A). CD34 and CD31 labeled intraretinal blood vessels (Figure 1B and C). No CD34 or CD31 immunoreactivity was detected in ridge spindle cells (Figure 1B and C). Scattered CD68+ cells (presumed resident retinal microglia) were present in the inner retina posterior to the ridge, often adjacent to blood vessels, but were found only rarely within the ridge (Figure 1D and E). Occasional lightly GFAP-immunoreactive cells were detected in the ridges (Figure 1F). Strongly GFAP-labeled astrocytes were detected in the optic nerve and throughout the vascularized inner retina (Figure 1G). Essentially all cells within ridge vanguards exhibited intense PAX2 immunoreactivity (Figure 1H and I). Desmin immunoreactivity was seen in extraocular muscles and around intraretinal arterioles but not in the ridge; neuron-specific enolase–labeled neurons were absent in the ridges (not shown).
Immunohistochemical staining of the ridge in eyes with retinopathy of prematurity. All sections oriented with anterior retina to the left. A, Low-power micrograph showing the thickened ridge vanguard (V), rearguard (R), and artifactual separation (*). Scale bar, 200 μm. PAX2-labeled cells were common posterior to the ridge but were infrequent anteriorly (open vs closed arrows). B, CD34+ vessels just posterior to the ridge; the ridge itself is negative (case 2) (original magnification ×20). C, CD31 labels posterior inner retinal vessels and extraretinal neovascularization (NV) (case 1) (original magnification ×20). D, Single CD68+ cell in the ridge (arrow) (case 2) (original magnification ×20). E, Several CD68+ cells exist in the inner retina, just posterior to the ridge, often adjacent to blood vessels (case 2) (original magnification ×20). F, Sparse lightly glial fibrillary acidic protein–immunoreactive cells (arrows) in the ridge (case 1) (original magnification ×20). G, Intense glial fibrillary acidic protein staining of inner retinal astrocytes in the posterior retina (case 2) (original magnification ×20). H, PAX2 labels essentially all ridge cells (case 2, left eye) (original magnification ×20). I, Intense PAX2 labeling of the ridge (case 2, right eye) (original magnification ×20).
Most endothelial cells lining rearguard vessels exhibited CD31 and/or CD34 immunoreactivity (Figure 2A and B). Several CD68+ presumed microglia were found among the vessels (Figure 2C). Rare GFAP+ and several PAX2+ glial cells were present in the rearguard (Figure 2D and E).
Immunohistochemical staining of the ridge rearguard. Asterisk indicates artifactual separation. Scale bar, 200 μm. A and B, CD31 (A) and CD34 (B) labeled endothelial cells in rearguard vascular structures. C, Several CD68+ cells scattered in the rearguard. D, Very few glial fibrillary acidic protein (GFAP)–positive cells (arrows). E, Several PAX2+ cells mostly surrounding the vascular structures. F, Rare Ki67 cells in the rearguard (arrows).
Ki-67+ cells appeared only rarely in the rearguard (Figure 2F). In case 1, Ki-67+ spindle cells were infrequently detected in ridge vanguards, while in case 2, numerous proliferating spindle cells were scattered throughout the ridges (Figure 3A and B). As a positive control, proliferating cells were found in extraretinal neovascular tufts and basal corneal epithelium (Figure 3C and D).
Cellular proliferation in ridge spindle cells. A and B, Ki-67 immunoreactivity in ridge cells (arrows) in cases 2 (A) (original magnification ×30) and 1 (B) (original magnification ×20). C and D, Ki67 labeled occasional cells in a tuft of neovascularization and in the inner retina (C) (arrows) (original magnification ×10) and basal corneal epithelium (D) (arrows) (original magnification ×20).
In normal retinal development, blood vessels arise at the optic nerve head and extend anteriorly to reach the periphery near term. Astrocytes similarly arise from the optic nerve and extend peripherally in advance of the nascent vasculature. In premature infants exposed to supplemental oxygen therapy, vessel growth arrests and a hypercellular ridge may develop at the border between vascularized and avascular retina. Although the ridge plays a critical role in ROP as the site of vascular shunting, and of either disease regression or progression, definitive description of the cellular composition of the ridge is lacking. Spindle-shaped ridge cells have been suggested to be mesenchymal angioblasts, or a heterogeneous population of vascular precursor cells, glia, and possibly pericyte precursors and accessory cells, that serve a transient developmental function.2 Two studies concluded that at least some ridge cells are glial, based on poorly defined microscopic features and GFAP labeling.3,4 We found that nearly all spindle-shaped cells that compose the ridge vanguard are glial: they are predominantly PAX2+ astrocyte precursors and, to a far lesser extent, mature GFAP+ astrocytes. We found no evidence of immature vascular endothelial cells (CD31+/CD34+) within the ridge vanguard. Only a few scattered CD68+ microglia were found in the ridges. Hypercellularity in the ridge vanguard may arise by cell division or by focal accumulation of astrocyte precursors whose radial migration is interrupted at the border of vascularized and avascular retina. The first possibility is supported by enhanced proliferation of retinal astrocyte precursors in low oxygen, while the second is supported by reduced astrocyte migration in hypoxia.5,6 Modest and variable proliferation among ridge cells and the scarcity of astrocyte precursors anterior to the ridge are consistent with both mechanisms. Resolution of this issue requires analysis of proliferation in retinae with late stage 1 and early stage 2 ROP, when the ridge begins to form. Animal models of ROP are of limited value, as they do not exhibit a ridge.
Astrocyte precursors that lie ahead of developing retinal vessels secrete vascular endothelial growth factor and other cytokines that appear to stimulate and guide peripheral extension of retinal vessels.5 It seems likely, then, that vascular endothelial growth factor secretion from ridge cells may stimulate neovascularization, which typically appears just posterior to the ridge. If this is the case, laser ablation of the ridge might be beneficial in cases where peripheral avascular retinal laser therapy is insufficient to cause regression of neovascular tissue. Interestingly, the exuberant dilated vascular tissue at the rearguard exhibited little or no cell proliferation, suggesting that either proliferation had given rise to these vessels earlier but had ceased by the time of death or that vessels there respond to vasoactive cytokines with dilation rather than frank neovascularization. In summary, ridge spindle cells consist mainly of astrocyte precursor cells and, to a lesser extent, mature astrocytes. Cell proliferation contributes at least partly to ridge formation.
Correspondence: Dr Gariano, Department of Ophthalmology, Room A-157, 300 Pasteur Dr, Stanford University School of Medicine, Palo Alto, CA 94305 (firstname.lastname@example.org).
Additional Contributions: Ed Gilbert, Stanford Immunopathology Laboratory, provided technical expertise. We thank Peter Egbert, MD, for helpful discussion.
Financial Disclosure: None reported.
Sun Y, Dalal R, Gariano RF. Cellular Composition of the Ridge in Retinopathy of Prematurity. Arch Ophthalmol. 2010;128(5):638-641. doi:10.1001/archophthalmol.2010.59